Manufacture of active highly phosphorylated human lysosomal sulfatase enzymes and uses thereof

ABSTRACT

This invention provides compositions of active highly phosphorylated lysosomal sulfatase enzymes, their pharmaceutical compositions, methods of producing and purifying such lysosomal sulfatase enzymes and compositions and their use in the diagnosis, prophylaxis, or treatment of diseases and conditions, including particularly lysosomal storage diseases that are caused by, or associated with, a deficiency in the lysosomal sulfatase enzyme.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/226,169, filed Jul. 16, 2009. This application isalso a continuation-in-part application of U.S. patent application Ser.No. 12/355,453 filed Jan. 16, 2009, which claims the benefit of U.S.Provisional Patent Application No. 61/022,179, filed Jan. 18, 2008, ofU.S. Provisional Patent Application No. 61/099,373, filed Sep. 23, 2008,and of U.S. Provisional Patent Application No. 61/110,246, filed Oct.31, 2008.

FIELD OF THE INVENTION

The present invention relates to the technical fields of cellular andmolecular biology and medicine, particularly to the manufacture ofactive highly phosphorylated human lysosomal sulfatase enzymes and theiruse in the management of the lysosomal storage diseases associated withlysosomal sulfatase enzyme deficiency. In particular, the presentinvention relates to the manufacture of active highly phosphorylatedrecombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and its usein the management of Mucopolysaccharidosis IVa (MPS IVa or Morquio Asyndrome) and other lysosomal storage diseases associated with adeficiency of GALNS.

BACKGROUND OF THE INVENTION

Lysosomal storage diseases (LSDs) result from the deficiency of specificlysosomal enzymes within the cell that are essential for the degradationof cellular waste in the lysosome. A deficiency of such lysosomalenzymes leads to accumulation within the lysosome of undegraded “storagematerial,” which causes swelling and malfunction of the lysosomes andultimately cellular and tissue damage. A large number of lysosomalenzymes have been identified and correlated with their related diseases.Once a missing enzyme has been identified, treatment can be reduced tothe sole problem of efficiently delivering a replacement enzyme to theaffected tissues of patients.

One way to treat lysosomal storage diseases is by intravenous enzymereplacement therapy (ERT) (Kakkis, Expert Opin. Investig. Drugs 11(5):675-685, 2002). ERT takes advantage of the vasculature to carry enzymefrom a single site of administration to most tissues. Once the enzymehas been widely distributed, it must be taken up into cells. The basisfor uptake into cells is found in a unique feature of lysosomal enzymes.Lysosomal enzymes constitute a separate class of glycoproteins definedby phosphate at the 6-position of terminal mannose residues.Mannose-6-phosphate is bound with high affinity and specificity by areceptor found on the surface of most cells (Munier-Lehmann et al.,Biochem. Soc. Trans. 24(1): 133-136, 1996; Marnell et al., J. Cell.Biol. 99(6): 1907-1916, 1984). The mannose-6-phosphate receptor (MPR),which has two mannose-6-phosphate binding sites per polypeptides chain(Tong et al., J. Biol. Chem. 264:7962-7969, 1989), directs uptake ofenzyme from blood to tissue and then mediates intracellular routing tothe lysosome.

Large-scale production of lysosomal enzymes involves expression inmammalian cell lines. The goal is the predominant secretion ofrecombinant enzyme into the surrounding growth medium for harvest andprocessing downstream. In an ideal system for the large-scale productionof lysosomal enzymes, enzyme would be efficiently phosphorylated andthen directed primarily toward the cell surface (i.e., for secretion),rather than primarily to the lysosome. As described above, thispartitioning of phosphorylated lysosomal enzymes is the exact oppositeof what occurs in normal cells. Manufacturing cell lines used forlysosomal enzyme production focuses on maximizing the level ofmannose-6-phosphate per mole of enzyme, but is characterized by lowspecific productivity. In vitro attempts at producing lysosomal enzymescontaining high levels of mannose-6-phosphate moieties have resulted inmixed success (Canfield et al., U.S. Pat. No. 6,537,785). The in vitroenzyme exhibits high levels of mannose-6-phosphate, as well as highlevels of unmodified terminal mannose. Competition between themannose-6-phosphate and mannose receptors for lysosomal enzyme resultsin the necessity for high doses of enzyme for effectiveness, and couldlead to greater immunogenicity to the detriment of the subject beingtreated.

Sulfatases constitute a unique subclass of lysosomal enzymes. Sulfatasescleave sulfate esters from a variety of substrates, including, forexample, steroids, carbohydrates, proteoglycans and glycolipids. Allknown eukaryotic sulfatases contain a cysteine residue at theircatalytic site. Sulfatase activity requires post-translationalmodification of this cysteine residue to C_(α)-formylglycine (FGly). Thecysteine to FGly post-translational enzyme activation occurs within theendoplasmic reticulum on unfolded sulfatases immediately aftertranslation, prior to targeting of the sulfatases to the lysosome(Dierks et al., Proc. Natl. Acad. Sci. USA 94:11963-11968, 1997). Theformylglycine-generating enzyme that catalyzes this reaction issulfatase modifying factor 1 (SUMF1). Highlighting the importance ofthis unique post-translational modification is the fact that mutationsin SUMF1, which result in impaired FGly formation in lysosomal sulfataseenzymes, cause Multiple Sulfatase Deficiency (MSD) in man (Diez-Ruiz etal., Annu. Rev. Genomics Hum. Genet. 6:355-379, 2005).

Accordingly, the therapeutic effectiveness of a lysosomal sulfataseenzyme preparation depends on the level of mannose-6-phosphate, and onthe presence of active enzyme, in that preparation.

Thus, there exists a need in the art for an efficient and productivesystem for the large-scale manufacture of therapeutically effective,active highly phosphorylated lysosomal sulfatase enzymes for managementof lysosomal storage disorders caused by or associated with a deficiencyof such lysosomal sulfatase enzymes.

SUMMARY OF INVENTION

The present invention relates to the discovery that when a CHO-K1 cellline derivative (designated G71) that is defective in endosomalacidification is engineered to express recombinant human sulfatasemodifying factor 1 (SUMF1), the modified G71 cells produce high yieldsof active highly phosphorylated recombinant lysosomal sulfatase enzymesin part by preventing loss of material to the lysosomal compartment ofthe manufacturing cell line. In one embodiment, the invention providesan END3 complementation group cell line that co-expresses recombinanthuman SUMF1 and recombinant human N-acetylgalactosamine-6-sulfatase(GALNS), resulting in high yields of active highly phosphorylatedenzyme. Exemplary cell lines are G71, G71S, and derivatives thereof,which retain the desired property of G71, i.e., the ability to producehigh yields of activate highly phosphorylated recombinant lysosomalsulfatase enzymes. This application of an END3 complementation groupmodified CHO-K1 cell line co-expressing recombinant human SUMF1 and arecombinant lysosomal sulfatase enzyme would be especially useful forthe manufacture of active highly phosphorylated lysosomal sulfataseenzymes to be used for management of lysosomal storage diseases byenzyme replacement therapy (ERT).

In a first aspect, the present invention features a novel method ofproducing active highly phosphorylated recombinant human lysosomalsulfatase enzymes or biologically active fragments, mutants, variants orderivatives thereof in an END3 complementation group CHO cell orderivative thereof in amounts that enable their therapeutic use. In abroad embodiment, the method comprises the steps of: (a) culturing aCHO-derived END3 complementation group cell or derivative thereof; (b)preparing a first mammalian expression vector capable of expressing theactive highly phosphorylated recombinant human lysosomal sulfataseenzyme or biologically active fragment, mutant, variant or derivativethereof in the CHO-derived END3 complementation group cell or derivativethereof; (c) preparing a second mammalian expression vector capable ofexpressing recombinant human sulfatase modifying factor 1 (SUMF1) orbiologically active fragment, mutant, variant or derivative thereof inthe CHO-derived END3 complementation group cell or derivative thereof;(d) transfecting the CHO-derived END3 complementation group cell orderivative thereof with the first and second expression vectors; (e)selecting and cloning of a transfectant of a CHO-derived END3complementation group cell or derivative thereof that expresses theactive highly phosphorylated recombinant human lysosomal sulfataseenzyme or biologically active fragment, mutant, variant or derivativethereof; and (f) optimizing a cell culture process method formanufacturing the highly phosphorylated recombinant human lysosomalsulfatase enzyme or biologically active fragment, mutant, variant orderivative thereof. The recombinant human lysosomal sulfatase enzyme isselected from the group consisting of arylsulfatase A (ARSA),arylsulfatase B (ARSB), iduronate-2-sulfatase (IDS),sulfamidase/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase(G6S) and N-acetylgalactosamine-6-sulfatase (GALNS).

The method involves the steps of transfecting a cDNA encoding all orpart of the lysosomal sulfatase enzyme and a cDNA encoding all or partof the human SUMF1 into a CHO-derived END3 complementation group cell orderivative thereof. In some embodiments, the first and second expressionvectors, which are capable of expressing the encoding the active highlyphosphorylated recombinant human lysosomal sulfatase enzyme and humanSUMF1, respectively, are transfected simultaneously into the CHO-derivedEND3 complementation group cell or derivative thereof. In someembodiments, the first and second expression vectors are transfectedinto the CHO-derived END3 complementation group cell or derivativethereof sequentially. In some embodiments, a cDNA encoding for afull-length human lysosomal sulfatase enzyme is used, whereas in otherembodiments a cDNA encoding for a biologically active fragment, mutant,variant or derivative thereof is used. In some embodiments, a cDNAencoding for a full-length human SUMF1 is used, whereas in otherembodiments a cDNA encoding for a biologically active fragment, mutant,variant or derivative thereof is used. In some embodiments, multipleexpression vectors are used to transfer the human lysosomal sulfataseenzyme and human SUMF1 cDNAs simultaneously or sequentially into theCHO-derived END3 complementation group cell or derivative thereof. Insome embodiments, a single expression vector is used to transfer thehuman lysosomal sulfatase enzyme and human SUMF1 cDNAs simultaneouslyinto the CHO-derived END3 complementation group cell or derivativethereof. In a preferred embodiment, the CHO-derived END3 complementationgroup cell or derivative thereof is a G71 cell line, a G71S cell line,or a G71 or G71S derivative.

In a preferred embodiment, the method comprises producing an activehighly phosphorylated recombinant human lysosomal sulfatase enzyme,e.g., arylsulfatase A (ARSA), arylsulfatase B (ARSB),iduronate-2-sulfatase (IDS), sulfamidase/heparin-N-sulfatase (SGSH),N-acetylglucosamine-sulfatase (G6S) or N-acetylgalactosamine-6-sulfatase(GALNS), from an END3 complementation group CHO cell line or derivativethereof. In a particularly preferred embodiment, the method comprisesproducing active highly phosphorylated recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) from an END3 complementationgroup CHO cell line or derivative thereof. An END3 complementation groupcell line is any modified CHO cell line that retains the properties ofan END3 complementation group cell line, such as defective endosomalacidification. In a preferred embodiment, the CHO-derived END3complementation group cell or derivative thereof is a G71 cell line, aG71S cell line, or a G71 or G71S derivative.

In a second aspect, the present invention provides an endosomalacidification-deficient mammalian cell line characterized by its abilityto produce active highly phosphorylated recombinant human lysosomalsulfatase enzymes in amounts that enable use of the lysosomal sulfataseenzyme therapeutically. In preferred embodiments, the invention providesCHO-K1-derived END3 complementation group cell lines, designated G71,G71S, or derivatives thereof, which are capable of producing high yieldsof active highly phosphorylated recombinant human lysosomal sulfataseenzymes, thereby enabling the large scale production of such therapeuticlysosomal sulfatase enzymes. In more preferred embodiments, the cellline expresses and secretes a recombinant human lysosomal sulfataseenzyme in amounts of at least about 0.5, preferably at least about 0.75,more preferably at least about 1.0, and even more preferably at leastabout 1.25 picograms/cell/day.

An END3 complementation group cell line is any modified CHO cell linethat retains the properties of an END3 complementation group cell line,such as defective endosomal acidification. In one embodiment, the END3complementation group CHO cell line is derived from G71 or a derivativethereof and comprises (a) an expression vector for recombinant humansulfatase modifying factor 1 (SUMF1) and (b) an expression vector for arecombinant human lysosomal sulfatase enzyme, wherein the recombinanthuman lysosomal sulfatase enzyme is selected from the group consistingof arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronate-2-sulfatase(IDS), sulfamidase/heparin-N-sulfatase (SGSH),N-acetylglucosamine-sulfatase (G6S) andN-acetylgalactosamine-6-sulfatase (GALNS). In a preferred embodiment,the END3 complementation group CHO cell line comprises the expressionvector for recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).In a more preferred embodiment, the END3 complementation group CHO cellline expresses and secretes recombinant human GALNS. In anotherpreferred embodiment, the END3 complementation group CHO cell line isselected from the group consisting of clone 4, clone 5, clone C6, cloneC2, clone C5, clone C7, clone C10, clone C11 and clone C30. In a morepreferred embodiment, the END3 complementation group CHO cell line isclone C2. In another preferred embodiment, the END3 complementationgroup CHO cell line is adapted to growth in suspension.

In a third aspect, the invention provides recombinant human lysosomalsulfatase enzymes produced in accordance with the methods of the presentinvention and thereby present in amounts that enable using the lysosomalsulfatase enzymes therapeutically. The lysosomal sulfatase enzymes maybe full-length proteins, or fragments, mutants, variants or derivativesthereof. In some embodiments, the lysosomal sulfatase enzyme orfragment, mutant, variant or derivative thereof according to theinvention may be modified as desired to enhance its stability orpharmacokinetic properties (e.g., PEGylation, mutagenesis, fusion,conjugation). In preferred embodiments, the enzyme is a human lysosomalsulfatase enzyme, a fragment of the human lysosomal sulfatase enzymehaving a biological activity of a native sulfatase enzyme, or apolypeptide that has substantial amino acid sequence homology with thehuman lysosomal sulfatase enzyme. In some embodiments, the lysosomalsulfatase enzyme is a protein of human or mammalian sequence, origin orderivation. In other embodiments, the lysosomal sulfatase enzyme is suchthat its deficiency causes a human disease, such as MetachromaticLeukodystrophy or MLD (i.e., arylsulfatase A (ARSA)), Maroteaux-Lamysyndrome or MPS VI (i.e., arylsulfatase B (ARSB)), Hunter syndrome orMPS II (i.e., iduronate-2-sulfatase (IDS)), Sanfilippo A syndrome or MPSIIIa (i.e., sulfamidase/heparin-N-sulfatase (SGSH)), Sanfilippo Dsyndrome or MPS IIId (i.e., N-acetylglucosamine-sulfatase (G6S)) andMorquio A syndrome or MPS IVa (i.e., N-acetylgalactosamine-6-sulfatase(GALNS)). In a particularly preferred embodiment, the lysosomalsulfatase enzyme is such that its deficiency causes Morquio A syndromeor MPS IVa (i.e., N-acetylgalactosamine-6-sulfatase (GALNS)). In anotherparticularly preferred embodiment, the lysosomal sulfatase enzyme issuch that its deficiency is associated with a human disease, such asMultiple Sulfatase Deficiency or MSD (i.e.,N-acetylgalactosamine-6-sulfatase (GALNS)).

The lysosomal sulfatase enzyme can also be of human or mammaliansequence origin or derivation. In yet other embodiments of theinvention, in each of its aspects, the lysosomal sulfatase enzyme isidentical in amino acid sequence to the corresponding portion of a humanor mammalian lysosomal sulfatase enzyme amino acid sequence. In otherembodiments, the polypeptide moiety is the native lysosomal sulfataseenzyme from the human or mammal. In other embodiments, the lysosomalsulfatase enzyme polypeptide is substantially homologous (i.e., at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical in amino acidsequence) over a length of at least about 25, 50, 100, 150, or 200 aminoacids, or the entire length of the polypeptide, to the native lysosomalsulfatase enzyme amino acid sequence of the human or mammalian enzyme.In some embodiments, the lysosomal sulfatase enzyme is humanN-acetylgalactosamine-6-sulfatase (GALNS). The amino acid sequence ofhuman GALNS is set forth in SEQ ID NO: 4, of which amino acids 27 to 522correspond to the secreted precursor protein. In some embodiments, thisGALNS enzyme comprises or consists of an amino acid sequence at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% identical to amino acids 27 to 522 of SEQ ID NO: 4, or a sequenceidentical to amino acids 27 to 522 of SEQ ID NO: 4. The GALNS enzymepreferably retains the catalytic site amino acids corresponding to theCys at position 53 of the secreted precursor protein (amino acid 79 ofSEQ ID NO: 4), which is capable of being converted toC_(α)-formylglycine. The GALNS enzyme may also retain other amino acidsin the active site cavity, including at least 1, 2, 3, 4, 5, 6, 7, 8 orall of the charged amino acids: Asp288, Asn289, Asp39, Asp54, His236,Lys140, His142, Lys310 and the α-helix: Arg83. Sukegawa, Human MolecularGenetics, 2000, Vol. 9, No. 9 1283-1290, incorporated herein byreference in its entirety, describes additional mutations that decreaseGALNS activity in patients and correlates the severity of variousmutations to their respective 3-dimensional location within the enzyme.In other embodiments, the subject to which the lysosomal sulfataseenzyme is to be administered is human.

In preferred embodiments, the lysosomal sulfatase enzyme is a highlyphosphorylated recombinant human lysosomal sulfatase enzyme produced byan endosomal acidification-deficient cell line, e.g., a CHO-derived END3complementation group cell line. An END3 complementation group cell lineis any modified CHO cell line that retains the properties of an END3complementation group cell line, such as defective endosomalacidification. In a preferred embodiment, the CHO-derived END3complementation group cell or derivative thereof is a G71 cell line, aG71S cell line, or a G71 or G71S derivative. Alternatively, thelysosomal sulfatase enzyme may be produced by any host cell, e.g., anyCHO cell or CHO cell-derived line, cultured under conditions that permitexpression and secretion of highly phosphorylated recombinant lysosomalsulfatase enzyme at relatively high yield, e.g., in amounts of at leastabout 0.5, at least about 0.75, at least about 1.0, or at least about1.25 picograms/cell/day.

In more preferred embodiments, the recombinant human lysosomal sulfataseenzyme has a high level of phosphorylated oligosaccharides (i.e.,greater than about 0.25, preferably greater than 0.5, and morepreferably greater than about 0.75 bis-phosphorylated oligomannosechains per protein chain).

In some embodiments, the invention provides a recombinant humanlysosomal sulfatase enzyme, e.g., GALNS, with a specified high level ofphosphorylated oligosaccharides. For example, the lysosomal sulfataseenzyme has from 0.5 to 1.0 bis-phosphorylated oligomannose chains permonomeric protein chain, or from 0.5 to 0.9 bis-phosphorylatedoligomannose chains per monomeric protein chain, or from 0.5 to 0.8bis-phosphorylated oligomannose chains per monomeric protein chain, orfrom 0.5 to 0.75 bis-phosphorylated oligomannose chains per monomericprotein chain, or from 0.54 to 0.75 bis-phosphorylated oligomannosechains per monomeric protein chain. Other similar ranges arecontemplated, e.g., at least 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65bis-phosphorylated oligomannose chains per monomeric protein chain, upto 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.98, or 1.0 bis-phosphorylatedoligomannose chains per monomeric protein chain, or any combination ofany of these numbers. In preferred embodiments, the enzyme is arecombinant human N-acetylgalactosamine-6-sulfatase (GALNS), e.g., ofSEQ ID NO: 4.

In some embodiments, the recombinant human lysosomal sulfatase enzymehas a high percentage (i.e., at least about 50%, 55%, 60%, or 65%,preferably at least about 70%, 75%, 80%, 85%, 90%, or 95%) of conversionof the active site cysteine residue to C_(α)-formylglycine (FGly). Inpreferred embodiments, the enzyme is an active recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) and the active site cysteineresidue is the Cys at position 53 (position 79 of SEQ ID NO: 4).

In particular embodiments, the recombinant human lysosomal sulfataseenzyme has a high level of phosphorylated oligosaccharides, e.g., any ofthe ranges or levels of bis-phosphorylated oligomannose chains permonomeric protein chain described herein, together with a highpercentage of conversion of the active site cysteine residue toC_(α)-formylglycine (FGly), e.g., any of the percentages describedherein. In preferred embodiments, the enzyme is an active highlyphosphorylated recombinant human N-acetylgalactosamine-6-sulfatase(GALNS), e.g., of SEQ ID NO: 4.

In any of the preceding embodiments, at least 97%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, or atleast 65% of the recombinant human lysosomal sulfatase enzyme, e.g.,GALNS (SEQ ID NO: 4), is in the precursor form as determined byCoomassie Blue staining when subjected to SDS-PAGE under reducingconditions or by SDS-capillary gel electrophoresis (SDS-CGE).

In addition, the lysosomal sulfatase enzyme, e.g., GALNS (SEQ ID NO: 4),optionally also exhibits a specific activity that is at least about 30%(e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold,30-fold, 40-fold or 50-fold) greater than the specific activity of acontrol lysosomal sulfatase enzyme of the same amino acid sequence thathas been produced in host cells (e.g., CHO cells or CHO-derived cells)that do not express recombinant human SUMF1.

In any of the preceding embodiments, the lysosomal sulfatase enzymedescribed, e.g., GALNS (SEQ ID NO: 4), exhibits a specific uptake(Kuptake) into fibroblasts that is about 0.1 to 10 nM, or about 0.1 to 7nM, or about 0.5 to 5 nM, or about 1 to 5 nM, or about 1 to 3.5 nM,about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about3.5 nM, or any combination of any of these numbers.

In any of the preceding embodiments, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, or at least about 80% of the recombinant humanlysosomal sulfatase enzyme, e.g., GALNS (SEQ ID NO: 4), binds amannose-6-phosphate receptor column.

According to this aspect, purified preparations of any of theseembodiments of the lysosomal sulfatase enzyme are provided in which thelysosomal sulfatase enzyme component, e.g., GALNS (SEQ ID NO: 4) has apurity of at least about 90%, 95%, 97%, 98%, or 99% as determined byCoomassie Blue staining when subjected to SDS-PAGE under non-reducingconditions or by another method of determining purity (e.g., SDS-PAGEunder reducing or non-reducing conditions followed by staining withCoomassie Blue or silver, or chromatographic separation by HPLC,including C4 reverse phase (RP) or C3 RP), or size exclusionchromatography (SEC)). In some embodiments, a significant amount of thelysosomal sulfatase enzyme component of the purified preparation is inthe secreted precursor form (e.g., at least 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% precursor), as determined by Coomassie Blue stainingwhen subjected to SDS-PAGE under reducing conditions or by anothermethod of detecting precursor (e.g., SDS-PAGE under reducing conditionswith Coomassie Blue or silver staining, or chromatographic separation byHPLC (e.g., C4 reverse phase (RP), C3 RP) or size exclusionchromatography (SEC), or a combination of electrophoretic separation andchromatographic separation, e.g., SDS-PAGE followed by capillary gelelectrophoresis (SDS-CGE))

In particular embodiments, the lysosomal sulfatase enzyme component ofthe purified preparation has a high level of phosphorylatedoligosaccharides, e.g., any of the ranges or levels ofbis-phosphorylated oligomannose chains per monomeric protein chaindescribed herein, together with a high percentage of conversion of theactive site cysteine residue to C_(α)-formylglycine (FGly), e.g., any ofthe percentages described herein. In more particular embodiments, thepurified preparation has a Kuptake as described herein.

In related aspects, the invention provides sterile compositionscontaining any of the lysosomal sulfatase enzyme or purifiedpreparations described herein, together with a sterile pharmaceuticallyacceptable diluent, carrier and/or excipient. Such sterile compositionsmay take the form of solutions or lyophilized powder, optionally invials, that may be reconstituted by the addition of sterile diluent.

In a fourth aspect, the invention provides a method to purifyrecombinant human lysosomal sulfatase enzymes produced by the methods ofthe present invention. In a preferred embodiment, lysosomal sulfataseenzymes are purified using a two-column process (dye-ligandchromatography, e.g., Blue-Sepharose, and anion exchange chromatography,e.g., SE Hi-Cap) comprising at least five purification steps: (1)filtering the harvest, i.e., culture medium from an END3 complementationgroup CHO cell line or derivative thereof that expresses human sulfatasemodifying factor 1 (SUMF1) and the recombinant human lysosomal sulfataseenzyme; (2) pH adjusting the filtered harvest to pH 4.5 (to induceprecipitation of contaminating proteins); (3) loading the pH-adjustedfiltered harvest onto a dye-ligand column, e.g., Blue-Sepharose column,washing the column and eluting the lysosomal sulfatase enzyme from thecolumn; (4) loading the eluate from the dye-ligand column onto an anionexchange column, e.g., SE Hi-Cap column, washing the column and elutingthe lysosomal sulfatase enzyme from the column; and (5) ultrafiltratingand diafiltrating the eluate from the anion exchange. Optionally, thefiltered harvest in step (1) is concentrated 10-20 fold byultrafiltration before adjusting the pH. Optionally, the ultrafiltratedand diafiltrated lysosomal sulfatase enzyme in step (5) is formulated ina formulation buffer. In a particularly preferred embodiment, thelysosomal enzyme is a recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS).

In another preferred embodiment, lysosomal sulfatase enzymes arepurified using a three-column process (capture chromatography, e.g.,anion exchange SE Hi-Cap; intermediate chromatography, e.g., dye-ligandCapto BlueZinc, Chelating Sepharose FF or Capto Adhere; and polishingchromatography, e.g., ToyoPearl Butyl 650M, Phenyl Sepharose Hi-Sub orPhenyl Sepharose Low-Sub) comprising at least five purification steps:(1) ultrafiltering the harvest, i.e., culture medium from an END3complementation group CHO cell line or derivative thereof that expresseshuman sulfatase modifying factor 1 (SUMF1) and the recombinant humanlysosomal sulfatase enzyme, by, e.g., Sartoon Cassettes, (10 kDa,Hydrosart); (2) pH adjusting the filtered harvest to pH 4.5 (to induceprecipitation of contaminating proteins); (3) loading the pH-adjustedfiltered harvest onto a capture column, e.g., Fractogel EMD SE Hi-CAP(M) anion exchange, washing the column and eluting the lysosomalsulfatase enzyme from the column; (4) loading the eluate from thecapture column onto an intermediate column, e.g., dye-ligand CaptoBlueZinc, Chelating Sepharose FF or Capto Adhere, washing the column andeluting the lysosomal sulfatase enzyme from the column; and (5) loadingthe eluate on a polishing column, e.g., ToyoPearl Butyl 650M, PhenylSepharose Hi-Sub or Phenyl Sepharose Low-Sub, washing the column andeluting the lysosomal enzyme from the column. The eluted lysosomalenzyme from step (5) is formulated in a formulation buffer. Optionally,the eluted lysosomal sulfatase enzyme from step (5) is ultrafiltratedand then formulated in a formulation buffer. Optionally, the lysosomalsulfatase enzyme from the column in step (4) is exposed to pH 3.5 forlow pH viral inactivation prior to loading onto the polishing column instep (5). In a particularly preferred embodiment, the lysosomal enzymeis a recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

In a fifth aspect, the invention provides a purified preparation ofactive highly phosphorylated recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) or biologically active mutant,variant or derivative thereof useful for treating a subject sufferingfrom a lysosomal storage disease that is caused by (e.g.,Mucopolysaccharidosis type IVa (MPS IVa) or Morquio A syndrome) orassociated with (e.g., Multiple Sulfatase Deficiency (MSD)) a deficiencyin the GALNS enzyme. In a preferred embodiment, the purified preparationof active highly phosphorylated recombinant human GALNS has a GALNSenzyme component characterized by: (a) has a purity of at least about90%, 95%, 97%, 98%, or 99% as determined by Coomassie Blue staining orsilver staining when subjected to SDS-PAGE under non-reducingconditions; (b) has at least about 80%, 85%, 90%, or 95% conversion ofthe cysteine residue at position 53 to C_(α)-formylglycine (FGly)(position 79 of SEQ ID NO: 4); (c) is N-linked glycosylated at theasparagine residues at positions 178 and 397, wherein some of theoligomannose chains attached to the asparagine residue at position 178are bis-phosphorylated; (d) from 0.5 to 1.0, or from 0.5 to 0.9, or from0.5 to 0.8, or from 0.5 to 0.75, or from 0.54 to 0.75 bis-phosphorylatedoligomannose chains per monomeric protein chain (e.g., at least 0.4,0.45, 0.5, 0.55, 0.6, or 0.65 and up to 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,0.98, or 1.0 bis-phosphorylated oligomannose chains per monomericprotein chain, or any combination of any of these numbers); and (e) atleast 65%, or at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95% or97%) of the GALNS enzyme is in the precursor form as determined byCoomassie Blue staining when subjected to SDS-PAGE under reducingconditions or by SDS-capillary gel electrophoresis (SDS-CGE). Inaddition, the GALNS enzyme may optionally also (f) exhibit a specificactivity that is at least about 30% (e.g., 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 2.5-fold, 3-fold, 4-fold,5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold or 50-fold) greaterthan the specific activity of a control GALNS enzyme of the same aminoacid sequence that has been produced in host cells (e.g., CHO cells orCHO-derived cells) that do not express recombinant human SUMF1.Optionally, the GALNS enzyme exhibits a specific uptake (Kuptake) intofibroblasts that is about 0.1 to 10 nM, or about 0.1 to 7 nM, or about0.5 to 5 nM, or about 1 to 5 nM, or about 1 to 3.5 nM, about 1 nM, about1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about 3.5 nM, or anycombination of any of these numbers.

The purified, active highly phosphorylated recombinant human GALNSconsists of a major band of about 55-60 kDa (i.e., precursor human GALNSbeing at least about 75%, or at least 80%, preferably at least about85%, more preferably at least about 90%, and even more preferably atleast about 95%, or at least 97% of the visible proteins) and minorbands at ˜39 kDa and ˜19 kDa (i.e., mature or processed human GALNSbeing less than about 25%, less than about 20%, preferably less thanabout 15%, more preferably less than about 10%, and even more preferablyless than about 5% or less than about 3% of the visible proteins) whensubjected to SDS-PAGE under reducing conditions. In a particularlypreferred embodiment, the purified, active highly phosphorylatedrecombinant human GALNS consists essentially of a single band of about55-60 kDa (i.e., precursor human GALNS) when subjected to SDS-PAGE underreducing conditions. In one embodiment, the purified, active highlyphosphorylated recombinant human GALNS is useful for treating MPS IVa orMorquio A syndrome. In one embodiment, the purified, active highlyphosphorylated recombinant human GALNS is useful for treating MSD.

In a sixth aspect, the invention provides a method of treating diseasescaused all or in part by deficiency, or are associated with adeficiency, of a lysosomal sulfatase enzyme. The method comprisesadministering a therapeutic recombinant human lysosomal sulfatase enzymeproduced by the methods of the present invention, wherein the lysosomalsulfatase enzyme binds to an MPR receptor and is transported across thecell membrane, enters the cell and is delivered to the lysosomes withinthe cell.

In one embodiment, the method comprises treating a subject sufferingfrom a deficiency of a lysosomal sulfatase enzyme comprisingadministering to the subject in need thereof a therapeutically effectiveamount of said lysosomal sulfatase enzyme, wherein said lysosomalsulfatase enzyme is a recombinant human lysosomal sulfatase enzyme orbiologically active fragment, mutant, variant or derivative thereofproduced by a CHO-derived END3 complementation group cell or aderivative thereof. In some embodiments, the method comprisesadministering a therapeutic recombinant human lysosomal sulfataseenzyme, or a biologically active fragment, mutant, variant or derivativethereof, alone or in combination with a pharmaceutically acceptablecarrier, diluent or excipient. Preferred embodiments include optimizingthe dosage to the needs of the subjects to be treated, preferablymammals and most preferably humans, to most effectively ameliorate thedeficiency of the lysosomal sulfatase enzyme.

Such therapeutic lysosomal sulfatase enzymes are particularly useful,for example, in the treatment of patients suffering from lysosomalstorage diseases caused by a deficiency of a lysosomal sulfatase enzyme,such as patients suffering from Metachromatic Leukodystrophy or MLD,Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome,Mucopolysaccharidosis type II (MPS II) or Hunter syndrome,Mucopolysaccharidosis type IIIa (MPS IIIa) or Sanfilippo A syndrome,Mucopolysaccharidosis type IIId (MPS IIId) or Sanfilippo D syndrome, andMucopolysaccharidosis type IVa (MPS IVa) or Morquio A syndrome. In aparticularly preferred embodiment, the lysosomal storage disease is MPSIVa or Morquio A syndrome and the lysosomal sulfatase enzyme isrecombinant human N-acetylgalactosamine-6-sulfatase (GALNS). In yetother embodiments, the invention also provides pharmaceuticalcompositions comprising the deficient lysosomal sulfatase enzyme causingthe lysosomal storage disease and a pharmaceutically acceptable carrier,diluent or excipient.

In another embodiment, the method comprises treating a subject sufferingfrom a lysosomal storage disease that is associated with a deficiency inone or more lysosomal sulfatase enzymes comprising administering to thesubject in need thereof a therapeutically effective amount of alysosomal sulfatase enzyme, wherein said lysosomal sulfatase enzyme is arecombinant human N-acetylgalactosamine-6-sulfatase (GALNS) orbiologically active fragment, mutant, variant or derivative thereofproduced by a CHO-derived END3 complementation group cell or aderivative thereof. In some embodiments, the method comprisesadministering therapeutic recombinant human GALNS enzyme or abiologically active fragment, mutant, variant or derivative thereofalone or in combination with a pharmaceutically acceptable carrier,diluent or excipient. In a particularly preferred embodiment, thelysosomal storage disease is Multiple Sulfatase Deficiency (MSD).

In particularly preferred embodiments, the CHO-derived END3complementation group cell or a derivative thereof is a G71 cell line, aG71S cell line or a G71 or G71S derivative thereof.

In still another embodiment, the present invention provides for a methodof enzyme replacement therapy by administering a therapeuticallyeffective amount of lysosomal sulfatase enzyme to a subject in need ofthe enzyme replacement therapy, wherein the cells of the patient havelysosomes which contain insufficient amounts of the lysosomal sulfataseenzyme to prevent or reduce damage to the cells, whereby sufficientamounts of the lysosomal sulfatase enzyme enter the lysosomes to preventor reduce damage to the cells. The cells may be within or without theCNS or need not be set off from the blood by capillary walls whoseendothelial cells are closely sealed to diffusion of an active agent bytight junctions.

In a particular embodiment, the invention provides compositions andpharmaceutical compositions comprising an active recombinant humanlysosomal sulfatase enzyme having a biological activity which isreduced, deficient, or absent in the target lysosome and which isadministered to the subject. Preferred active human lysosomal sulfataseenzymes include, but are not limited to, arylsulfatase A, arylsulfataseB, iduronate-2-sulfatase, sulfamidase/heparan-N-sulfatase,N-acetylglucosamine-6-sulfatase, and N-acetylgalactosamine-6-sulfatase.In a preferred embodiment, N-acetylgalactosamine-6-sulfatase is theactive recombinant human lysosomal sulfatase enzyme.

In a preferred embodiment, the invention provides a method of treating asubject suffering from MPS IVa or Morquio A syndrome, or MSD, byadministering to the subject a therapeutically effective amount of anyof the recombinant human N-acetylgalactosamine-6-sulfatase (GALNS),purified preparations and/or sterile compositions described herein.

In a more preferred embodiment, the invention provides a method oftreating a subject suffering from MPS IVa or Morquio A syndrome, or MSD,by administering to the subject a therapeutically effective amount ofrecombinant human N-acetylgalactosamine-6-sulfatase (GALNS) produced byEND3 complementation group cells, wherein the recombinant human GALNShas a high level of conversion of the active site cysteine residue toC_(α)-formylglycine (FGly) (i.e., at least about 50%, preferably atleast about 70%, more preferably at least about 90%, even morepreferably at least about 95% conversion), and high levels ofphosphorylation (i.e., greater than about 0.25, preferably greater than0.5, and more preferably greater than about 0.75 bis-phosphorylatedoligomannose chains per protein chain).

In a particularly preferred embodiment, the invention provides a methodof treating a subject suffering from MPS IVa or Morquio A syndrome, orMSD, by administering to the subject a therapeutically effective amountof a purified, active highly phosphorylated recombinant human GALNSpreparation that has a GALNS enzyme component that: (a) has a purity ofat least about 90%, 95%, 97%, 98% or 99% as determined by Coomassie Bluestaining or silver staining when subjected to SDS-PAGE undernon-reducing conditions; (b) has at least about 80%, 85%, 90% or 95%conversion of the cysteine residue at position 53 to C_(α)-formylglycine(FGly) (position 79 of SEQ ID NO: 4); (c) from 0.5 to 1.0, or from 0.5to 0.9, or from 0.5 to 0.8, or from 0.5 to 0.75, or from 0.54 to 0.75bis-phosphorylated oligomannose chains per monomeric protein chain(e.g., at least 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65 and up to 0.7, 0.75,0.8, 0.85, 0.9, 0.95, 0.98, or 1.0 bis-phosphorylated oligomannosechains per monomeric protein chain, or any combination of any of thesenumbers); and (d) at least 65%, or at least 70% (e.g., at least 75%,80%, 85%, 90%, 95% or 97%) of the GALNS enzyme is in the precursor formas determined by Coomassie Blue staining when subjected to SDS-PAGEunder reducing conditions or by SDS-capillary gel electrophoresis(SDS-CGE). In addition, the GALNS enzyme may optionally also (e) exhibita specific activity that is at least about 30% (e.g., 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 2.5-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold or50-fold) greater than the specific activity of a control GALNS enzyme ofthe same amino acid sequence that has been produced in host cells (e.g.,CHO cells or CHO-derived cells) that do not express recombinant humanSUMF1. Optionally, the GALNS enzyme exhibits a specific uptake (Kuptake)into fibroblasts that is about 0.1 to 10 nM, or about 0.1 to 7 nM, orabout 0.5 to 5 nM, or about 1 to 5 nM, or about 1 to 3.5 nM, about 1 nM,about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM or about 3.5 nM, orany combination of any of these numbers.

The purified, active highly phosphorylated recombinant human GALNSconsists of a major band of about 55-60 kDa (i.e., precursor human GALNSbeing at least about 75%, or at least about 80%, preferably at leastabout 85%, more preferably at least about 90%, and even more preferablyat least about 95% of the visible proteins) and minor bands at ˜39 kDaand ˜19 kDa (i.e., mature or processed human GALNS being less than about25%, or less than about 20%, preferably less than about 15%, morepreferably less than about 10%, and even more preferably less than about5% of the visible proteins) when subjected to SDS-PAGE under reducingconditions. In a more particularly preferred embodiment, the purified,active highly phosphorylated recombinant human GALNS consistsessentially of a single band of about 55-60 kDa (i.e., precursor humanGALNS) when subjected to SDS-PAGE under reducing conditions.

In some embodiments, the subject is suffering from MPS IVa or Morquio Asyndrome. In some embodiments, the subject is suffering from MSD.

Corresponding use of active highly phosphorylated lysosomal sulfataseenzymes of the invention, which are preferably produced by methods ofthe invention, in preparation of a medicament for the treatment of thelysosomal storage diseases described above is also contemplated.

In a seventh aspect, the present invention provides pharmaceuticalcompositions comprising an active highly phosphorylated recombinanthuman lysosomal sulfatase enzyme as described hereinabove which isuseful for treating diseases caused all or in part by, or are associatedwith, the deficiency in such lysosomal sulfatase enzyme, and one or morepharmaceutically acceptable carriers, diluents or excipients. In apreferred embodiment, the pharmaceutical composition comprises an activehighly phosphorylated recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) or biologically activefragment, mutant, variant or derivative thereof produced by the methodsof the invention and one or more pharmaceutically acceptable carriers,diluents or excipients. Such pharmaceutical compositions may be suitablefor administration by several routes such as intrathecal, parenteral,topical, intranasal, inhalational or oral administration. In a preferredembodiment, the pharmaceutical compositions are suitable for parenteraladministration. Within the scope of this aspect are embodimentsfeaturing nucleic acid sequences encoding the full-length lysosomalsulfatase enzymes or fragments, mutants, variants or derivativesthereof, which may be administered in vivo into cells affected with alysosomal enzyme deficiency.

In another aspect, the invention provides a method for detectingactivity of a lysosomal sulfatase enzyme comprising (a) culturingchondrocyte cells from a patient suffering from lysosomal sulfataseenzyme deficiency, e.g., a patient suffering from Morquio syndrome,under conditions that promote maintenance of chondrocytedifferentiation; (b) contacting the chondrocytes with a lysosomalsulfatase enzyme that degrades keratan sulfate; and (c) detecting levelsof keratan sulfate in the cells, wherein a reduced keratan sulfate levelin cells contacted with the lysosomal sulfatase enzyme compared to cellsnot contacted with the lysosomal sulfatase enzyme is indicative oflysosomal sulfatase enzyme activity. In some embodiments, the lysosomalsulfatase enzyme is N-acetylgalactosamine-6-sulfatase (GALNS). In someembodiments, the culturing is carried out in media comprising insulingrowth factor 1 (IGF1), transforming growth factor beta (TGF-β),transferrin, insulin and ascorbic acid. In some embodiments, the keratansulfate is detected by confocal microscopy, or via binding toanti-keratan sulfate antibody. The method may be carried out with anylysosomal sulfatase enzyme, including naturally occurring or recombinanthuman enzyme, or fragments or variants thereof, including variantscomprising an amino acid sequence at least 80%, 85%, 90%, 95% or 100%identical to the precursor human enzyme, without signal sequence, or themature form thereof.

In yet another aspect, the invention provides a cell-based assay formeasuring the activity of a recombinant human lysosomal enzyme todegrade natural substrates. The method comprises (a) culturing anisolated human cell deficient in the lysosomal enzyme under conditionsin which natural substrates for the lysosomal enzyme accumulate; (b)contacting the cell with the lysosomal enzyme; (c) lysing the cell; (d)adding to the cell lysate an enzyme that (i) is specific for the naturalsubstrates, and (ii) cleaves small oligosaccharides from the naturalsubstrates; (e) labeling the small oligosaccharides with a detectablemoiety; (f) optionally separating the labeled small oligosaccharides;(g) detecting the labeled small oligosaccharides; and (h) determiningthe activity of the lysosomal enzyme to degrade the natural substratesby comparing (i) the amount of labeled small oligosaccharide from cellscontacted with the lysosomal enzyme with (ii) the amount of labeledsmall oligosaccharides from cells not contacted with the lysosomalenzyme, wherein a reduction in (h)(i) as compared to (h)(ii) indicatesthe activity of the lysosomal enzyme to degrade natural substrates. Inone embodiment, the small oligosaccharide is a mono-, di, ortri-saccharide. In a related embodiment, the small oligosaccharide is adisaccharide. In some embodiments, the lysosomal enzyme is selected fromthe group consisting of arylsulfatase B (ARSB), iduronate-2-sulfatase(IDS), sulfamidase/heparin-N-sulfatase (SGSH),N-acetylglucosamine-sulfatase (G6S) andN-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, thelysosomal enzyme is α-L-iduronidase (IDU). In some embodiments, thelysosomal enzyme is acid α-glucosidase (GAA). In some embodiments, thelysosomal enzyme is β-glucoronidase (GUSB). In some embodiments, thelysosomal enzyme is β-galactosidase (GLB1).

Suitable human cells that can be used in the cell-based assay includeany human cell that is deficient in the lysosomal enzyme to be tested,such that can accumulate the natural substrates for the lysosomalenzyme. For example, cells naturally exhibiting a full (100%) or partialdeficiency in activity, e.g. 30%, 50%, 70%, 80%, 90%, 95% reduction ormore in activity, may be used. Cells expressing a mutant enzyme withdiminished activity, or cells derived from patients suffering from alysosomal storage disease, e.g. a mucopolysaccharidosis, may be used.Cells recombinantly altered to knockout or reduce lysosomal enzymeactivity, e.g. through introducing a mutation to the encoding gene orits promoter or other regulatory region, may be used. Cells treated toreduce lysosomal enzyme activity, e.g. treated with antisense or RNAi toreduce enzyme expression, may be used.

Suitable enzymes that cleave (digest) small oligosaccharides fromcarbohydrates and that are “specific for” (i.e. predominantly digest)the natural substrates of the lysosomal enzyme may be selected by thoseof ordinary skill in the art. For example, for detection of activity ofGALNS or GLB1 (enzymes that degrades keratan sulfate) the enzyme of step(d) may be Keratanase II or any enzyme that acts primarily on keratansulfate. As another example, for detection of IDU, ARSB, IDS or GUSB(enzymes that degrade dermatan sulfate), the enzyme of step (d) may beChondroitinase ABC or any enzyme that acts primarily on dermatansulfate. As another example, for detection of IDU, IDS, SGHS, G6S orGUSB (enzymes that degrade heparan sulfate), the enzyme of step (d) maybe Heparanase I or Heparanase II, or both. As yet another example, fordetection of GAA (an enzyme that degrades glycogen), the enzyme of step(d) may be α-amylase or any enzyme that acts primarily on glycogen.

This cell-based method is capable of great sensitivity in detectinglysosomal enzyme activity. In some embodiments, the lysosomal enzymeactivity is detectable when the concentration of lysosomal enzyme is aslow as about 10 nM, or about 5 nM, or about 1 nM, or about 0.75 nM, orabout 0.5 nM, or about 0.25 nM, or about 0.1 nM, or about 0.05 nM, orabout 0.01 nM, or about 0.005 nM, or about 1 pM, or about 0.5 pM.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes the nucleotide sequence of human sulfatase modifyingfactor 1 (SUMF1) (SEQ ID NO:1).

FIG. 2 describes the amino acid sequence of human sulfatase modifyingfactor 1 (SUMF1) (SEQ ID NO:2).

FIG. 3 describes the nucleotide sequence of humanN-acetylgalactosamine-6-sulfatase (GALNS) (SEQ ID NO:3).

FIG. 4 describes the amino acid sequence of humanN-acetylgalactosamine-6-sulfatase (GALNS) (SEQ ID NO:4). The signalpeptide of 26 amino acids at the N-terminus is absent in processedGALNS.

FIG. 5 depicts the structure and characteristics of processed humanN-acetylgalactosamine-6-sulfatase (GALNS).

FIGS. 6A-6B show the expression of humanN-acetylgalactosamine-6-sulfatase (GALNS) from G71S cells co-transfectedwith human sulfatase modifying factor 1 (SUMF1) and human GALNSexpression vectors. FIG. 6A shows the G71S clone screen for active GALNSin 96-wells. FIG. 6B shows the G71S clone GALNS productivity inpicograms per cell per day.

FIG. 7 illustrates a schematic of the WAVE bioreactor controller usedfor large-scale production of G71S cells expressing humanN-acetylgalactosamine-6-sulfatase (GALNS) and variants thereof.

FIG. 8 shows the stability of purified humanN-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity upon storageat 4° C. (diamonds) or at −70° C. (triangles).

FIGS. 9A-9B shows the purification of humanN-acetylgalactosamine-6-sulfatase (GALNS) by Blue Sepharose 6 Fast Flowchromatography (FIG. 9A) followed by Fractogel SE Hi-CAP chromatography(FIG. 9B). Purity is determined by Coomassie Blue staining of SDS-PAGE(left) and by Western blotting using an anti-GALNS (IVA) antibody(right).

FIG. 10 shows the purification of humanN-acetylgalactosamine-6-sulfatase (GALNS) byultrafiltration/diafiltration (UF/DF), Fractogel SE Hi-Capchromatography, Zn-chelating Sepharose chromatography and ToyoPearlButyl 650M chromatography. Purity is determined by Coomassie Bluestaining of SDS-PAGE (top left) and by Western blotting using ananti-GALNS antibody (top right), an anti-Cathepsin L antibody (bottomleft) and an anti-CHOP (Chinese Hamster Ovary cell proteins (bottomright).

FIG. 11 shows a dose dependent decrease in the amount of dermatansulfate substrate was observed in the IDU-treated GM01391 cells.

FIG. 12 shows a dose dependent decrease in the amount of dermatansulfate substrate was observed in the ARSB-treated GM00519 cells.

FIG. 13 shows the uptake of human N-acetylgalactosamine-6-sulfatase(GALNS), either unlabeled (circles) or conjugated with A488 (squares) orA555 (triangles), by cultured synoviocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of a method thatreconciles the need for large-scale manufacture of recombinant lysosomalsulfatase enzymes with the requirement of an active highlyphosphorylated lysosomal sulfatase enzyme product that is efficient intargeting lysosomes and hence is therapeutically effective.

The therapeutic effectiveness of a lysosomal enzyme preparation dependson the level of mannose-6-phosphate in that preparation. Phosphate isadded to the target glycoprotein by a post-translational modification inthe endoplasmic reticulum and early Golgi. Folded lysosomal enzymesdisplay a unique tertiary determinant that is recognized by anoligosaccharide modification enzyme. The determinant is composed of aset of specifically spaced lysines and is found on most lysosomalenzymes despite absence of primary sequence homology. The modificationenzyme, UDP-GlcNAc phosphotransferase, binds to the protein determinantand adds GlcNAc-1-phosphate to the 6-position of terminal mannoseresidues on oligosaccharides proximate to the binding site; a secondenzyme, phosphodiester α-GlcNAcase, then cleaves the GlcNAc-phosphatebond to give a mannose-6-phosphate terminal oligosaccharide (Canfield etal., U.S. Pat. No. 6,537,785). The purpose of the mannose-6-phosphatemodification is to divert lysosomal enzymes from the secretory pathwayto the lysosomal pathway within the cell. Mannose-6-phosphate-bearingenzyme is bound by the MPR in the trans Golgi and routed to the lysosomeinstead of the cell surface.

In addition to the presence of the mannose-6-phosphate marker onlysosomal enzyme oligosaccharides, lysosomal routing of enzymes dependson the acidification of trafficking endosomes emerging from the end ofthe trans Golgi stack. Chemical quenching of the acidic environmentwithin these endosomes with diffusible basic molecules results indisgorgement of the vesicular contents, including lysosomal enzymes,into the extracellular milieu (Braulke et al., Eur. J. Cell Biol. 43(3):316-321, 1987). Acidification requires a specific vacuolar ATPaseembedded within the membrane of the endosome (Nishi et al., Nat. Rev.Mol. Cell Biol. 3(2): 94-103, 2002). Failure of this ATPase is expectedto enhance the secretion of lysosomal enzymes at the expense oflysosomal routing. Manufacturing cell lines that carry defects in thevacuolar ATPase would be expected to prevent non-productive diversion ofphosphorylated recombinant enzyme to the intracellular lysosomalcompartment.

In 1984, Chinese hamster ovary (CHO) cell mutants specifically defectivein endosomal acidification were generated and characterized (Park etal., Somat. Cell Mol. Genet. 17(2): 137-150, 1991). CHO-K1 cells werechemically mutagenized and selected for survival at elevatedtemperatures in the presence of toxins. These toxins required endosomalacidification for the full expression of their lethality (Marnell etal., J. Cell. Biol. 99(6): 1907-1916, 1984). In the former study, acocktail of two toxins with different mechanisms of action was chosen toavoid selection of toxin-specific resistance. The principle is thatwhile the probability of serendipitous mutations that result inresistance to one particular toxin is small, the probability of twosimultaneous serendipitous mutations specific for two entirely differenttoxins is non-existent. Selections were carried out at elevatedtemperature to allow for temperature-sensitive mutations. This geneticscreen resulted in two mutants, one of which was designated G.7.1 (G71),that were resistant to toxins at elevated temperatures. The lesion inG71 was not due to the uptake or mechanism of action of the two toxins,but resulted from an inability of the clone to acidify endosomes atelevated temperatures. This inability was also evident at permissivetemperatures (34° C.), although to a lesser extent. G71 cells were alsofound to be auxotrophic for iron at elevated temperatures, despitenormal uptake of transferrin from the medium (Timchak et al., J. Biol.Chem. 261(30): 14154-14159, 1986). Since iron was released fromtransferrin only at low pH, auxotrophy for iron despite normaltransferrin uptake indicated a failure in endosomal acidification.Another study demonstrated that the acidification defect was manifestedprimarily in endosomes rather than lysosomes (Stone et al., J. Biol.Chem. 262(20): 9883-9886, 1987). The data on G71 were consistent withthe conclusion that a mutation resulted in the destabilization of thevacuolar ATPase responsible for endosomal acidification. Destabilizationwas most evident at elevated temperatures (39.5° C.) but was partiallyexpressed even at lower temperatures (34° C.). A study of thetrafficking of two endogenous lysosomal enzymes, cathepsin D andalpha-glucosidase, in G71 cells (Park et al., Somat. Cell Mol. Genet.17(2):137-150, 1991) showed that both enzymes were quantitativelysecreted at elevated temperatures, and glycosylation of the enzymes wasunaffected. The secretion of phosphorylated acid alpha-glucosidase wassignificantly enhanced at non-permissive temperatures.

The therapeutic effectiveness of a lysosomal sulfatase enzymepreparation not only depends on the level of mannose-6-phosphate, butalso depends on the presence of active enzyme in that preparation. Allknown sulfatases contain a cysteine residue at their catalytic site;this cysteine residue is post-translationally modified toC_(α)-formylglycine (FGly) to activate the enzyme. This cysteine to FGlypost-translational enzyme activation, which is catalyzed by sulfatasemodifying factor 1 (SUMF1), occurs within the endoplasmic reticulum onunfolded sulfatases immediately after translation, prior to targeting ofthe sulfatases to the lysosome (Dierks et al., Proc. Natl. Acad. Sci.USA 94:11963-11968, 1997). The importance of this uniquepost-translational modification is highlighted by the fact thatmutations in SUMF1, which result in impaired FGly formation in lysosomalsulfatase enzymes, cause Multiple Sulfatase Deficiency (MSD) in man(Diez-Ruiz et al., Annu. Rev. Genomics Hum. Genet. 6:355-379, 2005).

Thus, the ability of G71 cells, mutant CHO cells that are defective inendosomal acidification, to co-express recombinant human sulfatasemodifying enzyme (SUMF1) and a human lysosomal sulfatase enzyme providesa mechanism for the large-scale production of active highlyphosphorylated recombinant human lysosomal sulfatase enzymes useful forthe management of lysosomal storage disorders caused by or associatedwith a deficiency of such lysosomal sulfatase enzymes.

I. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991).

Each publication, patent application, patent, and other reference citedherein is incorporated by reference in its entirety to the extent thatit is not inconsistent with the present disclosure.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Allelic variant” refers to any of two or more polymorphic forms of agene occupying the same genetic locus. Allelic variations arisenaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (i.e., no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. “Allelic variants” also refer to cDNAs derived from mRNAtranscripts of genetic allelic variants, as well as the proteins encodedby them.

“Amplification” refers to any means by which a polynucleotide sequenceis copied and thus expanded into a larger number of polynucleotidemolecules, e.g., by reverse transcription, polymerase chain reaction,and ligase chain reaction.

A first sequence is an “antisense sequence” with respect to a secondsequence if a polynucleotide whose sequence is the first sequencespecifically hybridizes with a polynucleotide whose sequence is thesecond sequence.

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecontact surface characteristics are complementary to each other. A firstpolynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is identical to thenucleotide sequence of the polynucleotide binding partner of the secondpolynucleotide. Thus, the polynucleotide whose sequence 5′-TATAC-3′ iscomplementary to a polynucleotide whose sequence is 5′-GTATA-3′. Anucleotide sequence is “substantially complementary” to a referencenucleotide sequence if the sequence complementary to the subjectnucleotide sequence is substantially identical to the referencenucleotide sequence.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term “fragment” when used in reference to polypeptides refers topolypeptides that are shorter than the full-length polypeptide by virtueof truncation at either the N-terminus or C-terminus of the protein orboth, and/or by deletion of an internal portion or region of theprotein. Fragments of a polypeptide can be generated by methods known inthe art.

The term “mutant” when used in reference to polypeptides refers topolypeptides in which one or more amino acids of the protein have beensubstituted by a different amino acid. The amino acid substitution canbe a conservative substitution, as defined above, or can be anon-conservative substitution. Mutant polypeptides can be generated bymethods known in the art.

The term “derivative” when used in reference to polypeptides refers topolypeptides chemically modified by such techniques, for example and notfor limitation, as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation (i.e.,derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins. Derivative polypeptides can begenerated by methods known in the art.

The term “derivative” when used in reference to cell lines refers tocell lines that are descendants of the parent cell line; for example,this term includes cells that have been passaged or subcloned fromparent cells and retain the desired property, descendants of the parentcell line that have been mutated and selected for retention of thedesired property, and descendants of the parent cell line which havebeen altered to contain different expression vectors or differentexogenously added nucleic acids.

“Detecting” refers to determining the presence, absence, or amount of ananalyte in a sample, and can include quantifying the amount of theanalyte in a sample or per cell in a sample.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantitate the amount of bounddetectable moiety in a sample. The detectable moiety can be incorporatedin or attached to a primer or probe either covalently, or through ionic,van der Waals or hydrogen bonds, e.g., incorporation of radioactivenucleotides, or biotinylated nucleotides that are recognized bystreptavadin. The detectable moiety may be directly or indirectlydetectable. Indirect detection can involve the binding of a seconddirectly or indirectly detectable moiety to the detectable moiety. Forexample, the detectable moiety can be the ligand of a binding partner,such as biotin, which is a binding partner for streptavadin, or anucleotide sequence, which is the binding partner for a complementarysequence, to which it can specifically hybridize. The binding partnermay itself be directly detectable, for example, an antibody may beitself labeled with a fluorescent molecule. The binding partner also maybe indirectly detectable, for example, a nucleic acid having acomplementary nucleotide sequence can be a part of a branched DNAmolecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., Fahrlander et al.,Bio/Technology 6:1165, 1988). Quantitation of the signal is achieved by,e.g., scintillation counting, densitometry, or flow cytometry.

“Diagnostic” means identifying the presence or nature of a pathologiccondition. Diagnostic methods differ in their specificity andselectivity. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

The term “effective amount” means a dosage sufficient to produce adesired result on a health condition, pathology, and disease of asubject or for a diagnostic purpose. The desired result may comprise asubjective or objective improvement in the recipient of the dosage.“Therapeutically effective amount” refers to that amount of an agenteffective to produce the intended beneficial effect on health.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Equivalent dose” refers to a dose, which contains the same amount ofactive agent.

“Expression control sequence” refers to a nucleotide sequence in apolynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked thereto.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

“Highly phosphorylated,” “high level of phosphorylation” and “high levelof phosphorylated oligosaccharides” refer to preparations of lysosomalsulfatase enzymes in which at least 50% of the lysosomal sulfataseenzyme binds to the cation-independent mannose-6-phosphate receptorthrough phosphorylated oligosaccharides. Binding is furthercharacterized by sensitivity to competition with mannose-6-phosphate. Ahighly phosphorylated lysosomal sulfatase enzyme may also refer to alysosomal sulfatase enzyme with at least 0.25, preferably at least 0.5,and more preferably at least 0.75 bis-phosphorylated oligomannose chainsper protein chain. Alternatively, a highly phosphorylated lysosomalsulfatase enzyme (GALNS) may refer to an enzyme in which the specificuptake, Kuptake (the concentration of enzyme/ligand that yields half ofthe maximal uptake value), in fibroblasts is about 0.1 to 10 nM, orabout 0.1 to 7 nM, or about 0.5 to 5 nM, or about 1 to 5 nM, or about 1to 3.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3nM or about 3.5 nM, or any combination of any of these numbers.

“Bis-phosphorylated oligomannose chains” as used herein refers tomannose-containing oligosaccharide chains that are N-linked toasparagine residues in lysosomal sulfatase enzymes and comprise twomannose-6-phosphate residues. Typically, the bis-phosphorylatedoligomannose chains have 7 mannose residues, i.e., bis-phosphate mannose7 (BPM7), which are linked to two GlcNAc residues, which in turn arelinked to the asparagine residue in the lysosomal sulfatase enzyme.

“Active,” “activated” and “high level of activation” refer topreparations of lysosomal sulfatase enzymes in which at least 50%, 55%,60%, 65% preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of theprotein's active site cysteine residue has been post-translationallymodified to C_(α)-formylglycine (FGly). Alternatively, “active,”“activated” and “high level of activation” refer to preparations oflysosomal sulfatase enzymes which exhibit a specific activity that is atleast about 30% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold,15-fold, 20-fold, 30-fold, 40-fold or 50-fold) greater than the specificactivity of a control lysosomal sulfatase enzyme of the same amino acidsequence that has been produced in host cells (e.g., CHO cells orCHO-derived cells) that do not express recombinant human SUMF1. Asuitable control preparation of lysosomal sulfatase enzyme preferablyhas the same amino acid sequence as the highly active preparation, andis expressed by the same gene using the same promoter or regulatorysequence(s) in the same host cell, except that the host cell does notexpress recombinant human SUMF1, is produced under the same or similarculturing conditions including for the same time period of culture, andoptionally is purified to the same or similar extent as the highlyactive preparation.

“Active highly phosphorylated” refers to refers to preparations oflysosomal sulfatase enzymes in which at least 50%, preferably at least70%, more preferably at least 90%, and even more preferably at least 95%of the protein's active site cysteine residue has beenpost-translationally modified to C_(α)-formylglycine (FGly) and with atleast 0.25, preferably at least 0.5, and more preferably at least 0.75bis-phosphorylated oligomannose chains per protein chain.

The term “biologically active” refers to polypeptide (i.e., enzyme)fragments, mutants, variants or derivatives thereof that retain at leasta substantial amount (e.g., at least about 50%, preferably at leastabout 70%, and more preferably at least about 90%) of one or morebiological activities of the full-length polypeptide. When used inreference to a lysosomal sulfatase enzyme, a biologically activefragment, mutant, variant or derivative thereof retains at least asubstantial amount of sulfatase activity (i.e., cleavage of sulfateesters from its target substrates). When used in reference to sulfatasemodifying factor 1 (SUMF1), a biologically active fragment, mutant,variant or derivative thereof retains at least a substantial amount offormylglycine-generating activity (i.e., modification of a lysosomalsulfatase enzyme's active site cysteine residue to C_(α)-formylglycine(FGly)).

The term “purity” or “pure” when used in reference to polypeptidesrefers to the amount of the polypeptide being analyzed in comparison toany contaminating substances that can be detected using a particularmethod. For the recombinant lysosomal sulfatase enzymes of theinvention, “purity” may be determined by subjecting the sulfatase enzymepreparation to electrophoretic separation by SDS-PAGE under reducing ornon-reducing conditions followed by staining with Coomassie Blue orsilver, or by chromatographic separation by HPLC (e.g., C4 reverse phase(RP), C3 RP) or by any other chromatographic separation, e.g., sizeexclusion (SEC) and the like. Using any one of these methods, thepurified recombinant lysosomal sulfatase enzymes of the invention have apurity of at least about 80%, or at least about 85%, preferably at leastabout 90%, more preferably at least about 95%, and even more preferablyat least about 97%, 98% or 99%.

The term “precursor” or “precursor form” refers to the form ofrecombinant lysosomal sulfatase enzyme that is secreted from a mammaliancell, i.e., lacking the signal sequence, but lacking certainmodifications, e.g., internal cleavage of the proteins, which normallyoccur in the lysosome. The term “mature,” “mature form,” “processed” or“processed form” refers to the form of recombinant lysosomal sulfataseenzyme that normally exists in the lysosome. For the recombinantlysosomal sulfatase enzymes of the invention, the relative abundance of“precursor” or “precursor form” and “mature,” “mature form,” “processed”or “processed form” may be determined by subjecting the sulfatase enzymepreparation to electrophoretic separation by SDS-PAGE under reducingconditions followed by staining with Coomassie Blue or silver, or bychromatographic separation by HPLC (e.g., C4 reverse phase (RP), C3 RP)or by any other chromatographic separation, e.g., size exclusion (SEC)and the like, or a combination of electrophoretic separation andchromatographic separation, e.g., SDS-PAGE followed by capillary gelelectrophoresis (SDS-CGE). Using these methods, the purified recombinantlysosomal sulfatase enzymes of the invention consist of at least about65%, 70%, or 75%, preferably at least about 80% or 85%, more preferablyat least about 90%, and even more preferably at least about 95% or 97%“precursor” or “precursor form.” Alternatively, using these methods, thepurified recombinant lysosomal sulfatase enzymes of the inventionconsist of less than about 35%, 30% or 25%, preferably less than about20% or 15%, more preferably less than about 10%, and even morepreferably less than about 5% or 3% “mature,” “mature form,” “processed”or “processed form.” In some embodiments, only the “precursor” or“precursor form” is detected (i.e., the sulfatase enzyme preparationconsists essentially of a single detectable band when subjected toSDS-PAGE under reducing conditions or a single peak when analyzed byHPLC.

The terms “identical” or percent “identity,” in the context of two ormore polynucleotide or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

“Linker” refers to a molecule that joins two other molecules, eithercovalently, or through ionic, van der Waals or hydrogen bonds, e.g., anucleic acid molecule that hybridizes to one complementary sequence atthe 5′ end and to another complementary sequence at the 3′ end, thusjoining two non-complementary sequences.

“Low level of phosphorylation” or “low phosphorylation” refers to apreparation of lysosomal sulfatase enzymes in which the uptake intofibroblast cells has a half maximal concentration of greater than 10 nMor the fraction of lysosomal sulfatase enzymes that binds amannose-6-phosphate receptor column is less than about 25%.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in a subject animal, including humans and mammals. Apharmaceutical composition comprises a pharmacologically effectiveamount of a therapeutic lysosomal sulfatase enzyme and also comprisesone or more pharmaceutically acceptable carriers, diluents orexcipients. A pharmaceutical composition encompasses a compositioncomprising the active ingredient(s), and the inert ingredient(s) thatmake up the carrier, diluent or excipient, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a lysosomal sulfatase enzyme of the presentinvention and one or more pharmaceutically acceptable carriers, diluentsor excipients.

“Pharmaceutically acceptable carrier, diluent or excipient” refers toany of the standard pharmaceutical carriers, diluents, buffers, andexcipients, such as, for example and not for limitation, a phosphatebuffered saline solution, 5% aqueous solution of dextrose, andemulsions, such as an oil/water or water/oil emulsion, and various typesof wetting agents and/or adjuvants. Suitable pharmaceutical carriers,diluents or excipients and formulations are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).Preferred pharmaceutical carriers, diluents or excipients depend uponthe intended mode of administration of the active agent. Typical modesof administration include, for example and not for limitation, enteral(e.g., oral) or parenteral (e.g., subcutaneous, intramuscular,intravenous or intraperitoneal) injection; or topical, transdermal, ortransmucosal administration.

A “pharmaceutically acceptable salt” is a salt that can be formulatedinto a lysosomal sulfatase enzyme for pharmaceutical use including,e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides. Conventional notationis used herein to portray polypeptide sequences: the left-hand end of apolypeptide sequence is the amino-terminus; the right-hand end of apolypeptide sequence is the carboxyl-terminus.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe,” when used in reference to a polynucleotide, refers to apolynucleotide that is capable of specifically hybridizing to adesignated sequence of another polynucleotide. A probe specificallyhybridizes to a target complementary polynucleotide, but need notreflect the exact complementary sequence of the template. In such acase, specific hybridization of the probe to the target depends on thestringency of the hybridization conditions. Probes can be labeled with,e.g., chromogenic, radioactive, or fluorescent moieties and used asdetectable moieties.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology. The compounds ofthe invention may be given as a prophylactic treatment to reduce thelikelihood of developing a pathology or to minimize the severity of thepathology, if developed.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A host cell thatcomprises the recombinant polynucleotide is referred to as a“recombinant host cell.” The gene is then expressed in the recombinanthost cell to produce, e.g., a “recombinant polypeptide.” A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well.

“Hybridizing specifically to,” “specific hybridization,” or “selectivelyhybridize to” refers to the binding, duplexing, or hybridizing of anucleic acid molecule preferentially to a particular nucleotide sequenceunder stringent conditions when that sequence is present in a complexmixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and Northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2 “Overview of principles of hybridization and thestrategy of nucleic acid probe assays”, Elsevier, New York. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the Tm for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 2× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

A “subject” of diagnosis or treatment is a human or non-human animal,including a mammal or a primate.

The phrase “substantially homologous” or “substantially identical” inthe context of two nucleic acids or polypeptides, generally refers totwo or more sequences or subsequences that have at least 40%, 60%, 80%,90%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using one of the following sequence comparison algorithms or byvisual inspection. Preferably, the substantial identity exists over aregion of the sequences that is at least about 50 residues in length,more preferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues. In a most preferred embodiment, the sequences aresubstantially identical over the entire length of either or bothcomparison biopolymers.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol. 48:443, 1970, by the search for similarity method ofPearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360, 1987. The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300sequences, each of a maximum length of 5,000 nucleotides or amino acids.The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps. Another algorithm that isuseful for generating multiple alignments of sequences is Clustal W(Thompson et al., Nucleic Acids Research 22: 4673-4680, 1994).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410, 1990.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., J. Mol. Biol.215:403-410, 1990). These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787, 1993). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described herein.

“Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition means that about 80% to 90% or more of the macromolecularspecies present in the composition is the purified species of interest.The object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) if the composition consists essentially of a singlemacromolecular species. Solvent species, small molecules (<500 Daltons),stabilizers (e.g., BSA), and elemental ion species are not consideredmacromolecular species for purposes of this definition. In someembodiments, the lysosomal sulfatase enzymes of the invention aresubstantially pure or isolated. In some embodiments, the lysosomalsulfatase enzymes of the invention are substantially pure or isolatedwith respect to the macromolecular starting materials used in theirsynthesis. In some embodiments, the pharmaceutical composition of theinvention comprises a substantially purified or isolated therapeuticlysosomal sulfatase enzyme admixed with one or more pharmaceuticallyacceptable carriers, diluents or excipients.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of pathology for the purpose of diminishingor eliminating those signs or symptoms. The signs or symptoms may bebiochemical, cellular, histological, functional, subjective orobjective. The lysosomal sulfatase enzymes of the invention may be givenas a therapeutic treatment or for diagnosis.

“Therapeutic index” refers to the dose range (amount and/or timing)above the minimum therapeutic amount and below an unacceptably toxicamount.

“Treatment” refers to prophylactic treatment or therapeutic treatment ordiagnostic treatment.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of lysosomalsulfatase enzyme of the present invention calculated in an amountsufficient to produce the desired effect in association with one or morepharmaceutically acceptable carriers, diluents or excipients. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular lysosomal sulfatase enzyme employed and theeffect to be achieved, and the pharmacodynamics associated with eachlysosomal sulfatase enzyme in the host.

II. PRODUCTION OF LYSOSOMAL SULFATASE ENZYMES

In one aspect, the present invention features a novel method ofproducing active highly phosphorylated lysosomal sulfatase enzymes inamounts that enable therapeutic use of such enzymes. In general, themethod features transformation of a suitable cell line with the cDNAencoding for human sulfatase modifying factor 1 (SUMF1) or abiologically active fragment, mutant, variant or derivative thereof anda cDNA encoding full-length lysosomal sulfatase enzyme or a biologicallyactive fragment, mutant, variant or derivative thereof. Those of skillin the art may prepare expression constructs other than those expresslydescribed herein for optimal production of such lysosomal sulfataseenzymes in suitable transfected cell lines therewith. Moreover, skilledartisans may easily design fragments of cDNA encoding biologicallyactive fragments, variants, mutants or derivatives of the naturallyoccurring SUMF1 or lysosomal sulfatase enzymes that possess the same orsimilar biological activity to the naturally occurring full-lengthenzymes.

Host Cells

Host cells used to produce recombinant lysosomal sulfatase enzymes areendosomal acidification-deficient cell lines characterized by theirability to produce such lysosomal sulfatase enzymes in amounts thatenable use of the enzyme therapeutically. The invention provides aCHO-K1-derived, END3 complementation group cell line, designated G71.The invention also provides a G71 cell line that has been adapted forgrowth in serum-free suspension culture, designated G71S. The inventionalso provides derivatives of the G71 and G71S cell lines which have beensubcloned further or which contain different expression plasmids.

Cells that contain and express DNA or RNA encoding a recombinant proteinare referred to herein as genetically modified cells. Mammalian cellsthat contain and express DNA or RNA encoding the recombinant protein arereferred to as genetically modified mammalian cells. Introduction of theDNA or RNA into cells is by a known transfection method, such as, forexample and not for limitation, electroporation, microinjection,microprojectile bombardment, calcium phosphate precipitation, modifiedcalcium phosphate precipitation, cationic lipid treatment,photoporation, fusion methodologies, receptor mediated transfer, orpolybrene precipitation. Alternatively, the DNA or RNA can be introducedby infection with a viral vector. Methods of production for cells,including mammalian cells, which express DNA or RNA encoding arecombinant protein are described in co-pending patent applications U.S.Ser. No. 08/334,797, entitled “In Vivo Protein Production and DeliverySystem for Gene Therapy”, by Richard F Selden, Douglas A. Treco andMichael W. Heartlein (filed Nov. 4, 1994); U.S. Ser. No. 08/334,455,entitled “In Vivo Production and Delivery of Erythropoietin orInsulinotropin for Gene Therapy”, by Richard F Selden, Douglas A. Trecoand Michael W. Heartlein (filed Nov. 4, 1994) and U.S. Ser. No.08/231,439, entitled “Targeted Introduction of DNA Into Primary orSecondary Cells and Their Use for Gene Therapy”, by Douglas A. Treco,Michael W. Heartlein and Richard F Selden (filed Apr. 20, 1994). Theteachings of each of these applications are expressly incorporatedherein by reference in their entirety.

In preferred embodiments, the host cell used to produce recombinantlysosomal sulfatase enzymes is an endosomal acidification-deficient cellline characterized by its ability to produce such lysosomal sulfataseenzymes in amounts that enable use of the enzyme therapeutically. Inpreferred embodiments, the invention provides a CHO-K1-derived, END3complementation group cell line, designated G71, and a G71 cell linethat has been adapted for growth in serum-free suspension culture,designated G71S, which co-express human sulfatase modifying factor 1(SUMF1) and a recombinant lysosomal sulfatase enzyme, and are thuscapable of producing high yields of active highly phosphorylatedlysosomal sulfatase enzymes, as specified in “DEFINITIONS”, therebyenabling the large scale production of therapeutic lysosomal sulfataseenzymes. In most preferred embodiments, the G71 or G71S cell line, orderivative thereof, expresses and secretes recombinant lysosomalsulfatase enzymes in amounts of at least about 0.5, preferably at leastabout 0.75, more preferably at least about 1.0, and even more preferablyat least about 1.25 picograms/cell/day.

Vectors and Nucleic Acid Constructs

A nucleic acid construct used to express the recombinant protein, eitherhuman sulfatase modifying factor 1 (SUMF1) or lysosomal sulfatase enzymeor both, can be one which is expressed extrachromosomally (episomally)in the transfected mammalian cell or one which integrates, eitherrandomly or at a pre-selected targeted site through homologousrecombination, into the recipient cell's genome. A construct which isexpressed extrachromosomally comprises, in addition to recombinantprotein-encoding sequences, sequences sufficient for expression of theprotein in the cells and, optionally, for replication of the construct.It typically includes a promoter, recombinant protein-encoding DNA and apolyadenylation site. The DNA encoding the recombinant protein ispositioned in the construct in such a manner that its expression isunder the control of the promoter. Optionally, the construct may containadditional components such as one or more of the following: a splicesite, an enhancer sequence, a selectable marker gene under the controlof an appropriate promoter, an amplifiable marker gene under the controlof an appropriate promoter, and a matrix attachment region (MAR) orother element known in the art that enhances expression of the regionwhere it is inserted.

In those embodiments in which the DNA construct integrates into thecell's genome, it need include only the recombinant protein-encodingnucleic acid sequences. Optionally, it can include a promoter and anenhancer sequence, a polyadenylation site or sites, a splice site orsites, nucleic acid sequences which encode a selectable marker ormarkers, nucleic acid sequences which encode an amplifiable marker, amatrix attachment region (MAR) or other element known in the art thatenhances expression of the region where it is inserted, and/or DNAhomologous to genomic DNA in the recipient cell, to target integrationof the DNA to a selected site in the genome (to target DNA or DNAsequences).

Cell Culture Methods

Mammalian cells containing the recombinant protein-encoding DNA or RNAare cultured under conditions appropriate for growth of the cells andexpression of the DNA or RNA. Those cells which express the recombinantprotein can be identified, using known methods and methods describedherein, and the recombinant protein can be isolated and purified, usingknown methods and methods also described herein, either with or withoutamplification of recombinant protein production. Identification can becarried out, for example, through screening genetically modifiedmammalian cells that display a phenotype indicative of the presence ofDNA or RNA encoding the recombinant protein, such as PCR screening,screening by Southern blot analysis, or screening for the expression ofthe recombinant protein. Selection of cells which contain incorporatedrecombinant protein-encoding DNA may be accomplished by including aselectable marker in the DNA construct, with subsequent culturing oftransfected or infected cells containing a selectable marker gene, underconditions appropriate for survival of only those cells that express theselectable marker gene. Further amplification of the introduced DNAconstruct can be effected by culturing genetically modified mammaliancells under appropriate conditions (e.g., culturing genetically modifiedmammalian cells containing an amplifiable marker gene in the presence ofa concentration of a drug at which only cells containing multiple copiesof the amplifiable marker gene can survive).

Genetically modified mammalian cells expressing the recombinant proteincan be identified, as described herein, by detection of the expressionproduct. For example, mammalian cells expressing active highlyphosphorylated lysosomal sulfatase enzymes can be identified by asandwich enzyme immunoassay. The antibodies can be directed toward theactive agent portion.

Variants of Lysosomal Sulfatase Enzymes

In certain embodiments, active highly phosphorylated lysosomal sulfataseenzyme mutants or variants may be prepared and will be useful in avariety of applications in which active highly phosphorylated lysosomalsulfatase enzymes may be used. Amino acid sequence mutants or variantsof the polypeptide can be substitutional, insertional or deletionmutants or variants. Deletion mutants or variants lack one or moreresidues of the native protein that are not essential for function orimmunogenic activity. A common type of deletion mutant or variant is onelacking secretory signal sequences or signal sequences directing aprotein to bind to a particular part of a cell. Insertional mutants orvariants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,also called fusion proteins, are discussed below.

Variants may be substantially homologous or substantially identical tothe unmodified lysosomal sulfatase enzyme as set out above. Preferredvariants are those which are variants of an active highly phosphorylatedlysosomal sulfatase enzyme polypeptide that retains at least some of thebiological activity, e.g. sulfatase activity, of the lysosomal sulfataseenzyme. Other preferred variants include variants of a humanN-acetylgalactosamine-6-sulfatase polypeptide that retain at least someof the sulfatase activity of the humanN-acetylgalactosamine-6-sulfatase.

Substitutional mutants or variants typically exchange one amino acid ofthe wild-type polypeptide for another at one or more sites within theprotein, and may be designed to modulate one or more properties of thepolypeptide, such as, for example and not for limitation, stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

One aspect of the present invention contemplates generatingglycosylation site mutants or variants in which the O- or N-linkedglycosylation site of the lysosomal sulfatase enzyme has been mutated.Such mutants or variants will yield important information pertaining tothe biological activity, physical structure and substrate bindingpotential of the active highly phosphorylated lysosomal sulfataseenzyme. In particular aspects, it is contemplated that other mutants orvariants of the active highly phosphorylated lysosomal sulfatase enzymepolypeptide may be generated that retain the biological activity buthave increased or decreased substrate binding activity. As such,mutations of the active site or catalytic region are particularlycontemplated in order to generate protein mutants or variants withaltered substrate binding activity. In such embodiments, the sequence ofthe active highly phosphorylated lysosomal sulfatase enzyme is comparedto that of the other related enzymes and selected residues arespecifically mutated.

Numbering the amino acids of the mature protein from the putative aminoterminus as amino acid number 1, exemplary mutations that may be usefulinclude, for example, substitution of all or some of potentiallyglycosylated asparagines, including positions 178 and 397 of recombinanthuman N-acetylgalactosamine-6-sulfatase (GALNS) (see FIG. 5).

Substrate binding can be modified by mutations at/near the active siteof the lysosomal sulfatase enzyme. Taking into consideration suchmutations are exemplary, those of skill in the art will recognize thatother mutations of the enzyme sequence can be made to provide additionalstructural and functional information about this protein and itsactivity.

In order to construct mutants or variants such as those described above,one of skill in the art may employ well known standard technologies.Specifically contemplated are N-terminal deletions, C-terminaldeletions, internal deletions, as well as random and point mutagenesis.

N-terminal and C-terminal deletions are forms of deletion mutagenesisthat take advantage, for example, of the presence of a suitable singlerestriction site near the end of the C- or N-terminal region. The DNA iscleaved at the site and the cut ends are degraded by nucleases such asBAL31, exonuclease III, DNase I, and S1 nuclease. Rejoining the two endsproduces a series of DNAs with deletions of varying size around therestriction site. Proteins expressed from such mutant can be assayed forappropriate biological function, e.g. enzymatic activity, usingtechniques standard in the art, and described in the specification.Similar techniques may be employed for internal deletion mutants byusing two suitably placed restriction sites, thereby allowing aprecisely defined deletion to be made, and the ends to be religated asabove.

Also contemplated are partial digestion mutants. In such instances, oneof skill in the art would employ a “frequent cutter” that cuts the DNAin numerous places depending on the length of reaction time. Thus, byvarying the reaction conditions it will be possible to generate a seriesof mutants of varying size, which may then be screened for activity.

A random insertional mutation may also be performed by cutting the DNAsequence with a DNase I, for example, and inserting a stretch ofnucleotides that encode, 3, 6, 9, 12, etc., amino acids and religatingthe end. Once such a mutation is made the mutants can be screened forvarious activities presented by the wild-type protein.

Point mutagenesis also may be employed to identify with particularitywhich amino acid residues are important in particular activitiesassociated with lysosomal sulfatase enzyme biological activity. Thus,one of skill in the art will be able to generate single base changes inthe DNA strand to result in an altered codon and a missense mutation.

The amino acids of a particular protein can be altered to create anequivalent, or even an improved, second-generation molecule. Suchalterations contemplate substitution of a given amino acid of theprotein without appreciable loss of interactive binding capacity withstructures such as, for example, antigen-binding regions of antibodiesor binding sites on substrate molecules or receptors. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. Thus, variouschanges can be made in the DNA sequences of genes without appreciableloss of their biological utility or activity, as discussed below.

In making such changes, the hydropathic index of amino acids may beconsidered. It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte & Doolittle, J. Mol. Biol., 157(1):105-132, 1982, incorporatedherein by reference). Generally, amino acids may be substituted by otheramino acids that have a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein.

In addition, the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101,incorporated herein by reference, states that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with a biological property of theprotein. As such, an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalentand immunologically equivalent protein.

Exemplary amino acid substitutions that may be used in this context ofthe invention include but are not limited to exchanging arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine. Other such substitutionsthat take into account the need for retention of some or all of thebiological activity whilst altering the secondary structure of theprotein will be well known to those of skill in the art.

Another type of variant that is contemplated for the preparation ofpolypeptides according to the invention is the use of peptide mimetics.Mimetics are peptide-containing molecules that mimic elements of proteinsecondary structure. See, for example, Johnson et al., “Peptide TurnMimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapmanand Hall, New York (1993). The underlying rationale behind the use ofpeptide mimetics is that the peptide backbone of proteins exists chieflyto orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples described above, to engineer second generation moleculeshaving many of the natural properties of lysosomal sulfatase enzymes,but with altered and even improved characteristics.

Modified Glycosylation of Lysosomal Sulfatase Enzymes

Variants of an active highly phosphorylated lysosomal sulfatase enzymecan also be produced that have a modified glycosylation pattern relativeto the parent polypeptide, for example, deleting one or morecarbohydrate moieties, and/or adding one or more glycosylation sitesthat are not present in the native polypeptide.

Glycosylation is typically either N-linked or O-linked. N-linked refersto the attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. The presence of either of thesetripeptide sequences in a polypeptide creates a potential glycosylationsite. Thus, N-linked glycosylation sites may be added to a polypeptideby altering the amino acid sequence such that it contains one or more ofthese tripeptide sequences. O-linked glycosylation refers to theattachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used. O-linkedglycosylation sites may be added by inserting or substituting one ormore serine or threonine residues into the sequence of the originalpolypeptide.

Domain Switching

Various portions of lysosomal sulfatase enzyme proteins possess a greatdeal of sequence homology. Mutations may be identified in lysosomalsulfatase enzyme polypeptides that may alter its function. These studiesare potentially important for at least two reasons. First, they providea reasonable expectation that still other homologs, allelic variants andmutants of this gene may exist in related species, such as rat, rabbit,monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep and cat. Uponisolation of these homologs, variants and mutants, and in conjunctionwith other analyses, certain active or functional domains can beidentified. Second, this will provide a starting point for furthermutational analysis of the molecule as described above. One way in whichthis information can be exploited is in “domain switching.”

Domain switching involves the generation of recombinant molecules usingdifferent but related polypeptides. For example, by comparing thesequence of a lysosomal sulfatase enzyme, e.g.N-acetylgalactosamine-6-sulfatase, with that of a similar lysosomalsulfatase enzyme from another source and with mutants and allelicvariants of these polypeptides, one can make predictions as to thefunctionally significant regions of these molecules. It is possible,then, to switch related domains of these molecules in an effort todetermine the criticality of these regions to enzyme function andeffects in lysosomal storage disorders. These molecules may haveadditional value in that these “chimeras” can be distinguished fromnatural molecules, while possibly providing the same or even enhancedfunction.

Based on the numerous lysosomal sulfatase enzymes now being identified,further analysis of mutations and their predicted effect on secondarystructure will add to this understanding. It is contemplated that themutants that switch domains between the lysosomal sulfatase enzymes willprovide useful information about the structure/function relationships ofthese molecules and the polypeptides with which they interact.

Fusion Proteins

In addition to the mutations described above, the present inventionfurther contemplates the generation of a specialized kind of insertionalvariant known as a fusion protein. This molecule generally has all or asubstantial portion of the native molecule, linked at the N- orC-terminus, to all or a portion of a second polypeptide. For example,fusions typically employ leader sequences from other species to permitthe recombinant expression of a protein in a heterologous host. Anotheruseful fusion includes the addition of an immunologically active domain,such as an antibody epitope, to facilitate purification of the fusionprotein. Inclusion of a cleavage site at or near the fusion junctionwill facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expressionsystems that may be used in the present invention. Particularly usefulsystems include, but are not limited to, the glutathione S-transferase(GST) system (Pharmacia, Piscataway, N.J.), the maltose binding proteinsystem (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.),the 6×His system (Qiagen, Chatsworth, Calif.). These systems are capableof producing recombinant polypeptides bearing only a small number ofadditional amino acids, which are unlikely to affect the antigenicability of the recombinant polypeptide. For example, both the FLAGsystem and the 6×His system add only short sequences, both of which areknown to be poorly antigenic and which do not adversely affect foldingof the polypeptide to its native conformation. Another N-terminal fusionthat is contemplated to be useful is the fusion of a Met-Lys dipeptideat the N-terminal region of the protein or peptides. Such a fusion mayproduce beneficial increases in protein expression or activity.

A particularly useful fusion construct may be one in which an activehighly phosphorylated lysosomal sulfatase enzyme polypeptide or fragmentthereof is fused to a hapten to enhance immunogenicity of a lysosomalsulfatase enzyme fusion construct. This may be useful in the productionof antibodies to the active highly phosphorylated lysosomal sulfataseenzyme to enable detection of the protein. In other embodiments, afusion construct can be made which will enhance the targeting of thelysosomal sulfatase enzyme-related compositions to a specific site orcell.

Other fusion constructs including a heterologous peptide with desiredproperties, e.g., a motif to target the lysosomal sulfatase enzyme to aparticular organ, tissue, or cell type. In a preferred embodiment, afusion construct including a bone targeting peptide, e.g., 6 asparticacid residues (6×Asp or 6D) fused to a lysosomal sulfatase enzyme maytarget the enzyme to particular sites in bone.

Other fusion constructs including a heterologous polypeptide withdesired properties, e.g., an Ig constant region to prolong serumhalf-life or an antibody or fragment thereof for targeting also arecontemplated. Other fusion systems produce polypeptide hybrids where itis desirable to excise the fusion partner from the desired polypeptide.In one embodiment, the fusion partner is linked to the recombinantactive highly phosphorylated lysosomal sulfatase enzyme polypeptide by apeptide sequence containing a specific recognition sequence for aprotease. Examples of suitable sequences are those recognized by theTobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) orFactor Xa (New England Biolabs, Beverley, Mass.).

Derivatives

As stated above, a derivative refers to polypeptides chemically modifiedby such techniques as, for example and not for limitation,ubiquitination, labeling (e.g., with radionuclides or various enzymes),covalent polymer attachment such as pegylation (derivatization withpolyethylene glycol) and insertion or substitution by chemical synthesisof amino acids such as ornithine. Derivatives of the lysosomal sulfataseenzyme are also useful as therapeutic agents and may be produced by themethods of the invention.

Polyethylene glycol (PEG) may be attached to the lysosomal sulfataseenzyme produced by the methods of the invention to provide a longerhalf-life in vivo. The PEG group may be of any convenient molecularweight and may be linear or branched. The average molecular weight ofthe PEG will preferably range from about 2 kiloDaltons (“kDa”) to about100 kDa, more preferably from about 5 kDa to about 50 kDa, mostpreferably from about 5 kDa to about 10 kDa. The PEG groups willgenerally be attached to the lysosomal sulfatase enzymes of theinvention via acylation or reductive alkylation through a reactive groupon the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to areactive group on the protein moiety (e.g., an aldehyde, amino, or estergroup). Addition of PEG moieties to polypeptides of interest can becarried out using techniques well known in the art. See, e.g.,International Publication No. WO 96/11953 and U.S. Pat. No. 4,179,337.

Ligation of the lysosomal sulfatase enzyme polypeptide with PEG usuallytakes place in aqueous phase and can be easily monitored by reversephase analytical HPLC. The PEGylated peptides can be easily purified bypreparative HPLC and characterized by analytical HPLC, amino acidanalysis and laser desorption mass spectrometry.

Labels

In some embodiments, the therapeutic lysosomal sulfatase enzyme islabeled to facilitate its detection. A “label” or a “detectable moiety”is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, labels suitable for use in the present invention include, butare not limited to, radioactive labels (e.g., ³²P), fluorophores (e.g.,fluorescein), electron-dense reagents, enzymes (e.g., as commonly usedin an ELISA), biotin, digoxigenin, or haptens as well as proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thehapten or peptide, or used to detect antibodies specifically reactivewith the hapten or peptide.

Examples of labels suitable for use in the present invention include,but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold, colored glass or plastic beads (e.g.,polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the lysosomal sulfatase enzyme according to methods well known in theart. Preferably, the label in one embodiment is covalently bound to thelysosomal sulfatase enzyme using an isocyanate reagent for conjugationof an active agent according to the invention. In one aspect of theinvention, the bifunctional isocyanate reagents of the invention can beused to conjugate a label to a lysosomal sulfatase enzyme to form alabel lysosomal sulfatase enzyme conjugate without an active agentattached thereto. The label lysosomal sulfatase enzyme conjugate may beused as an intermediate for the synthesis of a labeled conjugateaccording to the invention or may be used to detect the lysosomalsulfatase enzyme conjugate. As indicated above, a wide variety of labelscan be used, with the choice of label depending on sensitivity required,ease of conjugation with the desired component of the lysosomalsulfatase enzyme, stability requirements, available instrumentation, anddisposal provisions. Non-radioactive labels are often attached byindirect means. Generally, a ligand molecule (e.g., biotin) iscovalently bound to the lysosomal sulfatase enzyme. The ligand thenbinds to another molecule (e.g., streptavidin), which is eitherinherently detectable or covalently bound to a signal system, such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound.

The lysosomal sulfatase enzymes of the invention can also be conjugateddirectly to signal-generating compounds, e.g., by conjugation with anenzyme or fluorophore. Enzymes suitable for use as labels include, butare not limited to, hydrolases, particularly phosphatases, esterases andglycosidases, or oxidotases, particularly peroxidases. Fluorescentcompounds, i.e., fluorophores, suitable for use as labels include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Further examples of suitablefluorophores include, but are not limited to, eosin, TRITC-amine,quinine, fluorescein W, acridine yellow, lissamine rhodamine, B sulfonylchloride erythroscein, ruthenium (tris, bipyridinium), Texas Red,nicotinamide adenine dinucleotide, flavin adenine dinucleotide, etc.Chemiluminescent compounds suitable for use as labels include, but arenot limited to, luciferin and 2,3-dihydrophthalazinediones, e.g.,luminol. For a review of various labeling or signal producing systemsthat can be used in the methods of the present invention, see U.S. Pat.No. 4,391,904.

Means for detecting labels are well known to those of skill in the art.Thus, for example, where the label is radioactive, means for detectioninclude a scintillation counter or photographic film, as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product.Colorimetric or chemiluminescent labels may be detected simply byobserving the color associated with the label. Other labeling anddetection systems suitable for use in the methods of the presentinvention will be readily apparent to those of skill in the art. Suchlabeled modulators and ligands can be used in the diagnosis of a diseaseor health condition.

In a preferred embodiment, the method comprises the step of producingactive highly phosphorylated lysosomal sulfatase enzymes from cell lineswith defects in endosomal trafficking. In a particularly preferredembodiment, the method comprises the step of producing active highlyphosphorylated recombinant human N-acetylgalactosamine-6-sulfatase(GALNS) from the CHO cell line G71, or a derivative thereof. Productionof lysosomal sulfatase enzymes such as, for example and not forlimitation, GALNS, comprises the steps of: (a) developing a G71 or G71derivative cell line that co-expresses a recombinant human lysosomalsulfatase enzyme, e.g., N-acetylgalactosamine-6-sulfatase (GALNS), andrecombinant human sulfatase modifying factor 1 (SUMF1); (b) culturinghuman lysosomal sulfatase enzyme and SUMF1 co-expressing cell lines; and(c) scaling up of the human lysosomal sulfatase enzyme and SUMF1co-expressing cell lines to bioreactor for production of lysosomalsulfatase enzymes. In preferred embodiments, the human lysosomalsulfatase enzyme, e.g., N-acetylgalactosamine-6-sulfatase (GALNS), andhuman SUMF1 cDNAs are subcloned into mammalian expression vectorsbasically as described herein below.

For cell line development, G71 or G71S, a G71 clone adapted for growthin serum-free suspension culture, was co-transfected with a human GALNSmammalian expression vector, a human SUMF1 mammalian expression vectorand a selectable marker gene, and stable transformants were selected.After a first round of subcloning of stable transfectants, cell lineswere selected using the fluorescent substrate and specificallydesignated. G71 or G71S cell lines were analyzed for cell-specificproductivity (pg of product/cell) in spinners with microcarriers or insuspension culture, respectively. The best producers of human GALNS wereidentified and scaled-up to bioreactor for production of pre-clinicalmaterial.

In another embodiment, the invention provides a cell-based assay formeasuring the activity of a recombinant human lysosomal enzyme todegrade natural substrates. The method comprises (a) culturing anisolated human cell deficient in the lysosomal enzyme under conditionsin which natural substrates for the lysosomal enzyme accumulate; (b)contacting the cell with the lysosomal enzyme; (c) lysing the cell; (d)adding to the cell lysate an enzyme that (i) is specific for the naturalsubstrates, and (ii) cleaves small oligosaccharides from the naturalsubstrates; (e) labeling the small oligosaccharides with a detectablemoiety; (f) optionally separating the labeled small oligosaccharides;(g) detecting the labeled small oligosaccharides; and (h) determiningthe activity of the lysosomal enzyme to degrade the natural substratesby comparing (i) the amount of labeled small oligosaccharide from cellscontacted with the lysosomal enzyme with (ii) the amount of labeledsmall oligosaccharides from cells not contacted with the lysosomalenzyme, wherein a reduction in (h)(i) as compared to (h)(ii) indicatesthe activity of the lysosomal enzyme to degrade natural substrates. Inone embodiment, the small oligosaccharide is a mono-, di, ortri-saccharide. In a related embodiment, the small oligosaccharide is adisaccharide.

In some embodiments, the lysosomal enzyme is selected from the groupconsisting of arylsulfatase B (ARSB), iduronate-2-sulfatase (IDS),sulfamidase/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase(G6S) and N-acetylgalactosamine-6-sulfatase (GALNS). In someembodiments, the lysosomal enzyme is α-L-iduronidase (IDU). In someembodiments, the lysosomal enzyme is acid α-glucosidase (GAA). In someembodiments, the lysosomal enzyme is β-glucoronidase (GUSB). In someembodiments, the lysosomal enzyme is β-galactosidase (GLB1).

Suitable human cells that can be used in the cell-based assay includeany human cell that is deficient in the lysosomal enzyme to be tested,such that can accumulate the natural substrates for the lysosomalenzyme. For example, cells naturally exhibiting a full (100%) or partialdeficiency in activity, e.g. 30%, 50%, 70%, 80%, 90%, 95% reduction ormore in activity, may be used. Cells expressing a mutant enzyme withdiminished activity, or cells derived from patients suffering from alysosomal storage disease, e.g. a mucopolysaccharidosis, may be used.Cells recombinantly altered to knockout or reduce lysosomal enzymeactivity, e.g. through introducing a mutation to the encoding gene orits promoter or other regulatory region, may be used. Cells treated toreduce lysosomal enzyme activity, e.g. treated with antisense or RNAi toreduce enzyme expression, may be used.

Suitable enzymes that cleave (digest) small oligosaccharides fromcarbohydrates and that are “specific for” (i.e. predominantly digest)the natural substrates of the lysosomal enzyme may be selected by thoseof ordinary skill in the art. For example, for detection of activity ofGALNS or GLB1 (enzymes that degrades keratan sulfate) the enzyme of step(d) may be Keratanase II or any enzyme that acts primarily on keratansulfate. As another example, for detection of IDU, ARSB, IDS or GUSB(enzymes that degrade dermatan sulfate), the enzyme of step (d) may beChondroitinase ABC or any enzyme that acts primarily on dermatansulfate. As another example, for detection of IDU, IDS, SGHS, G6S orGUSB (enzymes that degrade heparan sulfate), the enzyme of step (d) maybe Heparanase I or Heparanase II, or both. As yet another example, fordetection of GAA (an enzyme that degrades glycogen), the enzyme of step(d) may be α-amylase or any enzyme that acts primarily on glycogen.

This cell-based method is capable of great sensitivity in detectinglysosomal enzyme activity. In some embodiments, the lysosomal enzymeactivity is detectable when the concentration of lysosomal enzyme is aslow as about 10 nM, or about 5 nM, or about 1 nM, or about 0.75 nM, orabout 0.5 nM, or about 0.25 nM, or about 0.1 nM, or about 0.05 nM, orabout 0.01 nM, or about 0.005 nM, or about 1 pM, or about 0.5 pM.

III. PURIFICATION OF LYSOSOMAL SULFATASE ENZYMES

Bioreactor material containing recombinant human GALNS was 0.2 μmsterile filtered and kept at 4° C. The bioreactor material was eitherloaded onto a capture column directly, or concentrated 10- to 20-fold byultra-filtration prior to loading onto a capture column. The bioreactormaterial or concentrated bioreactor material was pH adjusted to pH 4.5and then loaded onto a Blue-Sepharose column, washed sequentially with20 mM acetate/phosphate, 50 mM NaCl, pH 4.5 and 20 mM acetate/phosphate,50 mM NaCl, pH 6.0 and eluted with 20 mM acetate/phosphate, 100 mM NaCl,pH 7.0. The Blue-Sepharose column eluate was then loaded onto FractogelSE Hi-Cap, washed sequentially with 20 mM acetate/phosphate, 50 mM NaCl,pH 5.0 and 20 mM acetate/phosphate, 50 mM NaCl, pH 5.5, and eluted with20 mM acetate/phosphate, 50-350 mM NaCl gradient, pH 5.5. The FractogelSE Hi-Cap eluate was formulated in 10 mM NaOAc, 1 mM NaH₂PO₄, 0.005%Tween-80, pH 5.5.

Alternatively, the bioreactor material containing recombinant humanGALNS was concentrated 20-fold by ultra-filtration prior to loading ontoa capture column. The concentrated bioreactor material was pH adjustedto pH 4.5, filtered and then loaded onto a Fractogel SE Hi-Cap column,washed sequentially with 10 mM acetate/phosphate, 50 mM NaCl, pH 4.5 and10 mM acetate/phosphate, 50 mM NaCl, pH 5.0, and eluted with 10 mMacetate/phosphate, 140 mM NaCl, pH 5.0. The Fractogel SE Hi-Cap columneluate was then adjusted to 500 mM NaCl, pH 7.0 and loaded ontoZn-chelating Sepharose (Zn-IMAC) column, washed with 10 mMacetate/phosphate, 125 mM NaCl, 10 mM imidazole, pH 7.0, and eluted with10 mM acetate/phosphate, 125 mM NaCl, 90 mM imidazole, pH 7.0. TheZn-chelating Sepharose (Zn-IMAC) column eluate was adjusted to pH 3.5for low pH viral inactivation, adjusted to 10 mM acetate/phosphate, 2MNaCl, pH 5.0, and then loaded onto a ToyoPearl Butyl 650M column, washedwith 10 mM acetate/phosphate, 2M NaCl, pH 5.0, and eluted with 10 mMacetate/phosphate, 0.7 M NaCl, pH 5.0. The ToyoPearl Butyl 650M eluatewas ultra-filtrated and dia-filtrated in 20 mM acetate, 1 mM phosphate,150 mM NaCl, pH 5.5, and then formulated in 20 mM acetate, 1 mMphosphate, 150 mM NaCl, 0.01% Tween-20, pH 5.5.

The purification of recombinant human GALNS is described in detailinfra, and purification of recombinant human GALNS following proceduresmodified from the above protocol is described in detail infra.

Recombinant human GALNS enzyme was expressed in G71S cells as describedin Example III and purified as described in Example V. The purifiedrecombinant human GALNS of the invention can be compared to otherdocumented preparations of GALNS. Masue et al., J. Biochem. 110:965-970,1991 described the purification and characterization of GALNS from humanplacenta. The purified enzyme was found to have a molecular mass of 120kDa, consisting of polypeptides of 40 kDa and 15 kDa, the latter ofwhich was shown to be a glycoprotein. Thus, the Masue et al. GALNSenzyme appears to correspond to the processed form depicted in FIG. 5.Bielicki et al., Biochem. J. 279:515-520, 1991 described thepurification and characterization of GALNS from human liver. Whenanalysed by SDS-PAGE, the enzyme had a molecular mass of 70 kDa undernon-reducing conditions and molecular masses 57 kDa, 39 kDa and 19 kDaunder reducing conditions. Bielicki et al., Biochem J. 311: 333-339,1995 described the purification and characterization of recombinanthuman GALNS from Chinese hamster ovary cells. The purified enzyme onSDS-PAGE was found to have a molecular mass of 58-60 kDa undernon-reducing conditions and molecular masses of 55-57 kDa, 39 kDa and 38kDa under reducing conditions. Thus, the Bielicki et al. GALNS enzymesappear to correspond to a mixture of the pre-processed (precursor) formof the enzyme and the processed form depicted in FIG. 5. In contrast,the recombinant human GALNS enzyme of the invention consists almostentirely of the precursor form of the enzyme (see FIG. 9), orpredominantly (i.e., at least about 85%) of the precursor form of theenzyme (see FIG. 10).

IV. LYSOSOMAL SULFATASE ENZYMES AND LYSOSOMAL STORAGE DISEASES

The lysosomal sulfatase enzyme is a full-length enzyme or any fragment,mutant, variant or derivative thereof that retains at least asubstantial amount (e.g., at least about 50%, preferably at least about75%, and more preferably at least about 90%), substantially all, or allof the therapeutic or biological activity (e.g., sulfatase activity) ofthe enzyme.

In some embodiments, the lysosomal sulfatase enzyme is one that, if notexpressed or produced, or if substantially reduced in expression orproduction, would give rise to a disease, including but not limited to,lysosomal storage diseases. In some embodiments, the lysosomal sulfataseenzyme is one that, if not expressed or produced, or if substantiallyreduced in expression or production, may not give rise to a disease, butwhose absence or reduced expression or production is associated with thedisease, including but not limited to, lysosomal storage diseases.Preferably, the lysosomal sulfates enzyme is derived or obtained from ahuman.

Preferably, in the treatment of lysosomal storage diseases, thelysosomal sulfatase enzyme is an enzyme that is found in a cell that ifnot expressed or produced or is substantially reduced in expression orproduction, would give rise to a lysosomal storage disease.Alternatively, in the treatment of lysosomal storage diseases, thelysosomal sulfatase enzyme is an enzyme whose absence or substantiallyreduced expression or production is associated with the disease,although its absence or substantially reduced expression or production,may not itself give rise to the disease. Preferably, the lysosomalsulfatase enzyme is derived or obtained from a human.

Preferably, the enzyme is a lysosomal sulfatase enzyme, such asarylsulfatase A (ARSA) (Genbank Accession No. NP_(—)000478 (isoform a),Genbank Accession No. NP_(—)001078897 (isoform b) and other variants),arylsulfatase B/N-acetylglucosamine 4-sulfatase (ARSB) (GenbankAccession No. P15848), iduronate-2-sulfatase (IDS) (Genbank AccessionNo. NP_(—)000193 (isoform a), Genbank Accession No. NP_(—)006114(isoform b)), sulfamidase/heparin-N-sulfatase (SGSH) (Genbank AccessionNo. NP_(—)000190), N-acetylglucosamine-sulfatase (G6S) (GenbankAccession No. NP_(—)002067) and Galactose6-sulfatase/N-acetylgalactosamine-6-sulfatase (GALNS) (Genbank AccessionNo. NP_(—)000503). A table of lysosomal storage diseases and thelysosomal sulfatase enzymes deficient therein, which are useful astherapeutic agents, follows:

Lysosomal Storage Disease Lysosomal Sulfatase DeficiencyMucopolysaccharidosis type II Iduronate-2-sulfatase Hunter syndromeMucopolysaccharidosis type IIIA Sulfamidase/heparin-N-sulfataseSanfilippo syndrome Mucopolysaccharidosis type IIID N-Acetylglucosamine6-sulfatase Sanfilippo syndrome Mucopolysaccharidosis type IVAN-Acetylgalactosamine-6- Morquio syndrome sulfataseMucopolysaccharidosis type VI N-Acetylgalactosamine 4- sulfataseMetachromatic leukodystrophy (MLD) Arylsulfatase A Multiple sulfatasedeficiency (MSD) Multiple sulfatases

In preferred embodiments, the lysosomal sulfatase enzyme is arecombinant human lysosomal sulfatase enzyme produced by an endosomalacidification-deficient cell line. In more preferred embodiments, therecombinant human lysosomal sulfatase enzyme is active and has a highlevel of phosphorylated oligosaccharides as specified under“DEFINITIONS”. In most preferred embodiments, the lysosomal sulfataseenzyme is an active highly phosphorylated recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS).

Thus, the lysosomal storage diseases that can be treated or preventedusing the methods of the present invention include, but are not limitedto, Metachromatic Leukodystrophy or MLD, Maroteaux-Lamy syndrome or MPSVI, Hunter syndrome or MPS II, Sanfilippo A syndrome or MPS IIIa,Sanfilippo D syndrome or MPS IIId, and Morquio A syndrome or MPS IVa. Ina particularly preferred embodiment, the lysosomal sulfatase enzyme issuch that its deficiency causes Morquio A syndrome or MPS IVa. Inanother particularly preferred embodiment, the lysosomal sulfataseenzyme is such that its deficiency is associated with a human lysosomalstorage disease, such as Multiple Sulfatase Deficiency or MSD.

Thus, per the above table, for each disease the lysosomal sulfataseenzyme would preferably comprise a specific active lysosomal sulfataseenzyme deficient in the disease. For instance, for methods involving MPSII, the preferred enzyme is iduronate-2-sulfatase. For methods involvingMPS IIIA, the preferred enzyme is sulfamidase/heparin-N-sulfatase. Formethods involving MPS IIID, the preferred enzyme is N-acetylglucosamine6-sulfatase. For methods involving MPS IVA, the preferred enzyme isgalactose 6-sulfatase/N-acetylgalactosamine-6-sulfatase. For methodsinvolving MPSVI, the preferred enzyme is N-acetylgalactosamine4-sulfatase. For methods involving Metachromatic Leukodystrophy (MLD),the preferred enzyme is arylsulfatase A. For methods involving MultipleSulfatase Deficiency (MSD), the enzyme can be arylsulfatase A,arylsulfatase B/N-acetylglucosamine 4-sulfatase, iduronate-2-sulfatase,sulfamidase/heparin-N-sulfatase, N-acetylglucosamine-sulfatase orgalactose 6-sulfatase/N-acetylgalactosamine-6-sulfatase, and thepreferred enzyme is galactose6-sulfatase/N-acetylgalactosamine-6-sulfatase.

V. MUCOPOLYSACCHARIDOSIS TYPE IVA Morquio Syndrome, MPS IVa

Mucopolysaccharidosis type IVA (Morquio Syndrome, MPS IVa) is aninherited, autosomal recessive disease belonging to the group ofmucopolysaccharide storage diseases. Morquio Syndrome is caused by adeficiency of a lysosomal enzyme required for the degradation of twoglycosaminoglycans (GAGs), keratan sulfate (KS) andchondroitin-6-sulfate (C6S). Specifically, MPS IVa is characterized bythe absence of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS), andthe excretion of KS in the urine. The lack of GALNS results inaccumulation of abnormally large amounts of mucopolysaccharides inhyaline cartilage, a main component of skeletal tissues. All patientshave a systemic skeletal dysplasia. Other symptoms vary in severity frompatient to patient, and may include hearing loss, cataracts, spinalinstability, heart valvular disease and respiratory issues, amongothers.

GALNS hydrolyses sulfate ester bonds of galactose-6-sulfate from KS andN-acetylgalactosamine-6-sulfate from C6S. Human GALNS is expressed as a55-60 kDa precursor protein with only 2 potential asparagine-linkedglycosylation sites. Mannose-6-phosphate (M6P) is part of theoligosaccharides present on the GALNS molecule. M6P is recognized by areceptor at the lysosomal cell surface and, consequently, is crucial forefficient uptake of GALNS.

Like all sulfatases, GALNS needs to be processed by aformylglycine-activating enzyme (FGE) encoded by the sulfatase modifyingfactor1 (SUMF1) gene to gain activity. Because of this activation step,involving the post-translational modification of an active site cysteineresidue to C_(α)-formylglycine (FGly), over-expression of recombinantsulfatases can lead to both production of sulfatase enzymes with lowspecific activity (i.e., a mix of activated and non-activated sulfataseenzymes) and with low production titer (i.e., degradation and/ornon-secretion of non-activated sulfatases).

An object of this invention is to provide an active highlyphosphorylated human N-acetylgalactosamine-6-sulfatase enzyme useful forthe treatment of Morquio Syndrome and other diseases, e.g., MultipleSulfatase Deficiency (MSD), that are caused by or associated with adeficiency in the enzyme N-acetylgalactosamine-6-sulfatase. Such anactive highly phosphorylated human N-acetylgalactosamine-6-sulfataseenzyme has the ability to localize to tissues in which KS and C6Saccumulates, has adequate M6P levels for efficient uptake, hassufficiently high percentage of FGly in for enzyme activity, and hasrelatively high production levels.

It should be understood that the methods of the invention describedherein are applicable to the production of other lysosomal sulfataseenzymes, e.g., arylsulfatase A (ARSA), arylsulfataseB/N-acetylglucosamine 4-sulfatase (ARSB), iduronate-2-sulfatase (IDS),sulfamidase/heparin-N-sulfatase (SGSH) and N-acetylglucosamine-sulfatase(G6S), useful for the treatment of lysosomal storage diseases which arecaused or characterized by their deficiency thereof.

VI. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

The lysosomal sulfatase enzymes of the invention may be administered bya variety of routes. For oral preparations, the lysosomal sulfataseenzymes can be used alone or in combination with appropriate additivesto make tablets, powders, granules or capsules, for example, withconventional additives, such as lactose, mannitol, corn starch or potatostarch; with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch or gelatins; with disintegrators, suchas corn starch, potato starch or sodium carboxymethylcellulose; withlubricants, such as talc or magnesium stearate; and if desired, withdiluents, buffering agents, moistening agents, preservatives andflavoring agents.

The lysosomal sulfatase enzymes of the invention can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

The lysosomal sulfatase enzymes of the invention can be utilized inaerosol formulation to be administered via inhalation. The lysosomalsulfatase enzymes of the invention can be formulated into pressurizedacceptable propellants such as dichlorodifluoromethane, propane,nitrogen and the like.

Furthermore, the lysosomal sulfatase enzymes of the invention can bemade into suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. The lysosomal sulfataseenzymes of the invention can be administered rectally via a suppository.The suppository can include vehicles such as cocoa butter, carbowaxesand polyethylene glycols, which melt at body temperature, yet aresolidified at room temperature.

Unit dosage forms of the lysosomal sulfatase enzymes of the inventionfor oral or rectal administration such as syrups, elixirs, andsuspensions may be provided wherein each dosage unit, for example,teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of a lysosomal sulfatase enzyme containing activeagent. Similarly, unit dosage forms for injection or intravenousadministration may comprise the lysosomal sulfatase enzyme as a solutionin sterile water, normal saline or another pharmaceutically acceptablecarrier.

In practical use, the lysosomal sulfatase enzymes of the invention canbe combined as the active ingredient in intimate admixture with one ormore pharmaceutically acceptable carriers, diluents or excipientsaccording to conventional pharmaceutical compounding techniques. Thecarrier, diluent or excipient may take a wide variety of forms dependingon the preferable form of preparation desired for administration, e.g.,oral or parenteral (including intravenous). In preparing the lysosomalsulfatase enzyme compositions for oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like in the case of oral liquid preparations, forexample, suspensions, elixirs and solutions; or carriers such asstarches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like in thecase of oral solid preparations, for example, powders, hard and softcapsules and tablets, with the solid oral preparations being preferredover the liquid preparations.

With respect to transdermal routes of administration, methods fortransdermal administration of drugs are disclosed in Remington'sPharmaceutical Sciences, 17th Edition, (Gennaro et al., Eds. MackPublishing Co., 1985). Dermal or skin patches are a preferred means fortransdermal delivery of the lysosomal sulfatase enzymes of theinvention. Patches preferably provide an absorption enhancer such asDMSO to increase the absorption of the lysosomal sulfatase enzymes.Other methods for transdermal drug delivery are disclosed in U.S. Pat.Nos. 5,962,012, 6,261,595, and 6,261,595, each of which is incorporatedby reference in its entirety.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are also commercially available.

In each of these aspects, the lysosomal sulfatase enzyme compositionsinclude, but are not limited to, compositions suitable for oral, rectal,topical, parenteral (including subcutaneous, intramuscular, andintravenous), pulmonary (nasal or buccal inhalation), or nasaladministration, although the most suitable route in any given case willdepend in part on the nature and severity of the conditions beingtreated and on the nature of the active ingredient. Exemplary routes ofadministration are the oral and intravenous routes. The lysosomalsulfatase enzyme compositions may be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or non-aqueous techniques. The percentageof an active lysosomal sulfatase enzyme in these compositions may, ofcourse, be varied and may conveniently be between about 2 percent toabout 60 percent of the weight of the unit.

Lysosomal sulfatase enzyme compositions of the invention may beadministered encapsulated in or attached to viral envelopes or vesicles,or incorporated into cells. Vesicles are micellular particles which areusually spherical and which are frequently lipidic. Liposomes arevesicles formed from a bilayer membrane. Suitable vesicles include, butare not limited to, unilamellar vesicles and multilamellar lipidvesicles or liposomes. Such vesicles and liposomes may be made from awide range of lipid or phospholipid compounds, such asphosphatidylcholine, phosphatidic acid, phosphatidylserine,phosphatidylethanolamine, sphingomyelin, glycolipids, gangliosides, etc.using standard techniques, such as those described in, e.g., U.S. Pat.No. 4,394,448. Such vesicles or liposomes may be used to administerlysosomal sulfatase enzymes intracellularly and to deliver lysosomalsulfatase enzymes to the target organs. Controlled release of alysosomal sulfatase enzyme of interest may also be achieved usingencapsulation (see, e.g., U.S. Pat. No. 5,186,941).

Any route of administration that dilutes the lysosomal sulfatase enzymecomposition into the blood stream, or preferably, at least outside ofthe blood-brain barrier, may be used. Preferably, the lysosomalsulfatase enzyme composition is administered peripherally, mostpreferably intravenously or by cardiac catheter. Intrajugular andintracarotid injections are also useful. Lysosomal sulfatase enzymecompositions may be administered locally or regionally, such asintraperitoneally, subcutaneously or intramuscularly. In one aspect,lysosomal sulfatase enzyme compositions are administered with one ormore pharmaceutically acceptable carrier, diluent or excipient.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific lysosomal sulfatase enzyme, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given lysosomal sulfatase enzyme are readilydeterminable by those of skill in the art by a variety of meansincluding, but not limited to, dose response and pharmacokineticassessments conducted in patients, in test animals and in vitro.

Dosages to be administered may also depend on individual needs, on thedesired effect, the particular lysosomal sulfatase enzyme used, and onthe chosen route of administration. Dosages of a lysosomal sulfataseenzyme range from about 0.2 pmol/kg to about 20 nmol/kg, preferreddosages range from 2 pmol/kg to 2 nmol/kg, and particularly preferreddosages range from 2 pmol/kg to 200 pmol/kg. Alternatively, dosages ofthe lysosomal sulfatase enzyme may be in the range of 0.01 to 1000mg/kg, preferred dosages may be in the range of 0.1 to 100 mg/kg, andparticularly preferred dosages range from 0.1 to 10 mg/kg. These dosageswill be influenced by, for example and not for limitation, theparticular lysosomal sulfatase enzyme, the form of the pharmaceuticalcomposition, the route of administration, and the site of action of theparticular lysosomal sulfatase enzyme.

The lysosomal sulfatase enzymes of the invention are useful fortherapeutic, prophylactic and diagnostic intervention in animals, and inparticular in humans. Lysosomal sulfatase enzymes may show preferentialaccumulation in particular tissues. Preferred medical indications fordiagnostic uses include, for example, any condition associated with atarget organ of interest (e.g., lung, liver, kidney, spleen).

The subject methods find use in the treatment of a variety of differentdisease conditions. In certain embodiments, of particular interest isthe use of the subject methods in disease conditions where a lysosomalsulfatase enzyme having desired activity has been previously identified,but in which the lysosomal sulfatase enzyme is not adequately deliveredto the target site, area or compartment to produce a fully satisfactorytherapeutic result. With such lysosomal sulfatase enzymes, the subjectmethods of producing active highly phosphorylated lysosomal sulfataseenzymes can be used to enhance the therapeutic efficacy and therapeuticindex of the lysosomal sulfatase enzyme.

Treatment is meant to encompass any beneficial outcome to a subjectassociated with administration of a lysosomal sulfatase enzyme includinga reduced likelihood of acquiring a disease, prevention of a disease,slowing, stopping or reversing, the progression of a disease or anamelioration of the symptoms associated with the disease conditionafflicting the host, where amelioration or benefit is used in a broadsense to refer to at least a reduction in the magnitude of a parameter,e.g., symptom, associated with the pathological condition being treated,such as inflammation and pain associated therewith. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g., terminated, such that the host nolonger suffers from the pathological condition, or at least no longersuffers from the symptoms that characterize the pathological condition.

A variety of hosts or subjects are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

Having now generally described the invention, the same may be morereadily understood through the following reference to the followingexamples, which provide exemplary protocols for the production, andpurification of active highly phosphorylated lysosomal sulfatase enzymesand their use in the treatment of lysosomal storage diseases. Theexamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Effortshave been made to ensure accuracy with respect to numbers used (e.g.,amounts, temperatures, etc.), but some experimental error and deviationshould, of course, be allowed for.

EXAMPLES Example I Mammalian Expression Vectors for Human SulfataseModifying Factor1 (SUMF1) and Human N-Acetylgalactosamine-6-Sulfatase(GALNS)

The objective was to construct mammalian expression vectors appropriatefor producing in stably transfected cells adequate amounts of activelysosomal sulfatase enzymes with improved phosphorylation levels.

The full-length human sulfatase modifying factor 1 (SUMF1) cDNA (seeUnited States Patent Application Nos. US 20005/0123949, publication dateJun. 9, 2005, and US 2004/0229250, publication date Nov. 8, 2004, bothof which are herein incorporated by reference in their entirety), whichencodes a 374 amino acid polypeptide, was cloned into the mammalianexpression vector cDNA4 (Invitrogen, Carlsbad, Calif.), which containsthe human CMV enhancer-promoter and a multiple cloning site. Efficienttranscript termination was ensured by the presence of the bovine growthhormone polyadenylation sequence. The selection marker was a zeocinresistance gene under the control of the EM-7 promoter and SV40 earlypolyadenylation sequence. The resultant plasmid was designated pcDNA4SUMF1. The human SUMF1 polynucleotide (SEQ ID NO:1) and polypeptide (SEQID NO:2) sequences are shown in FIG. 1 and FIG. 2, respectively.

The full-length human N-acetylgalactosamine-6-sulfatase (GALNS) cDNA(see Tomatsu et al., Biochem. Biophys. Res. Commun. 181(2):677-683,1991), which encodes a 522 amino acid polypeptide including a 26 aminoacid signal peptide, was cloned into the mammalian expression vectorpCIN (BioMarin), which contains the human CMV enhancer-promoter linkedto the rabbit β-globin IVS2 intron and a multiple cloning site.Efficient transcript termination was ensured by the presence of thebovine growth hormone polyadenylation sequence. The selection marker wasa neomycin phosphotransferase gene that carries a point mutation todecrease enzyme efficiency. The attenuated marker was furtherhandicapped with the weak HSV-tk promoter. The resultant plasmid wasdesignated pCIN 4A. The human GALNS polynucleotide (SEQ ID NO:3) andpolypeptide (SEQ ID NO:4) sequences are shown in FIG. 3 and FIG. 4,respectively.

To increase the expression levels of SUMF1 and GALNS, scaffold/matrixattachment region (MAR) elements (see Mermod et al., U.S. Pat. No.7,129,062) were cloned into the SUMF1 and GALNS expression plasmids.

BMAR SUMF1 was made by digesting P<1_(—)68 X_X NcoI filled MAR (Selexis)with BamHI and HincII, and then inserting the released MAR fragment intopcDNA4 SUMF1 digested with BglII and NruI.

PMAR SUMF1 was made digesting P<1_(—)68 NcoI filled (MAR) SV40 EGFP(Selexis) with HindIII and XbaI to remove the EGFP gene, and theninserting the SUMF1 gene, which was released from pcDNA4 SUMF1 bydigestion with HindIII and XbaI.

BMAR 4A was made by digesting BMAR SUMF1 with PmeI and SpeI to removethe SUMF1 gene, and then inserting the GALNS gene, which was releasedfrom pCIN 4A by digestion with PmeI and SpeI.

PMAR 4A was made by digesting P<1_(—)68 NcoI filled (MAR) SV40 EGFP(Selexis) with HindIII and XbaI to remove the EGFP gene, and theninserting the GALNS gene, which was released from pCIN 4A by digestionwith HindIII and XbaI.

The full-length human GALNS cDNA was also cloned into the mammalianexpression vector pcDNA4 (Invitrogen, Carlsbad, Calif.). pcDNA4 SUMF1was digested with HindIII and XbaI to remove the SUMF1 cDNA, and pCIN 4Awas digested with HindIII and XbaI to isolate the GALNS cDNA. The GALNScDNA HindIII/XbaI fragment was ligated into the pcDNA4 vectorHindIII/XbaI fragment. The resultant plasmid was designated pcDNA4-4A.

The integrity of the GALNS gene in the pCIN 4A, BMAR and pcDNA4-4Aexpression vectors was confirmed by restriction mapping using enzymesobtained from New England Biolabs. The PMAR 4A expression vector was notmapped.

The structure of the fully processed form of humanN-acetylgalactosamine-6-sulfatase (GALNS) is depicted in FIG. 5. GALNSis expressed as a 522 amino acid polypeptide with a 26 amino acid signalpeptide sequence. A 496 amino acid GALNS polypeptide is secreted as apre-processed (precursor) form of the enzyme having a molecular weightof about 55-60 kDa. In active GALNS, the cysteine residue at position 53of the precursor or fully processed GALNS polypeptide (corresponding toposition 79 of the full-length GALNS polypeptide) has been converted toC_(α)-formylglycine (FGly) by sulfatase modifying factor 1 (SUMF1). Inthe lysosome, GALNS is cleaved after position 325 of the fully processedGALNS polypeptide, resulting in GALNS peptide fragments of about 40 kDaand 19 kDa. These GALNS peptides are joined by a disulfide bridgebetween the cysteine (C) residues at positions 282 and 393 of the fullyprocessed GALNS polypeptide. There are two canonical N-linkedglycosylation sites, at positions 178 and 397 of the fully processedGALNS polypeptide. Bis-phosphorylated mannose 7 (BPM7), comprising 2mannose-6-phosphate residues, has been found on N178, but not on N397.

Example II G71S Cell Lines Co-Expressing Human Sulfatase ModifyingFactor1 (SUMF1) and Human N-Acetylgalactosamine-6-Sulfatase (GALNS)

The objective was to develop cell lines capable of producing activelysosomal sulfatase enzymes with improved phosphorylation levels.

G71 cells (Rockford K. Draper) were derived directly from CHO-K1 (ATCCCCL-61). The G71 cell line is a temperature-sensitive mutant of CHO-K1with respect to acidification of the endosomes, which has been observedto yield differences in total protein secretion and phosphorylation onmannose residues for several enzymes at elevated temperatures (Park etal., Somat. Cell Mol. Genet. 17(2): 137-150, 1991; Marnell et al., J.Cell. Biol. 99(6): 1907-1916, 1984).

G71 cells were maintained at 34° C. in BioWhittaker UltraCHO mediumsupplemented with 2.5% fetal calf serum, 2 mM glutamine, gentamycin andamphotericin.

To allow easier use of cell lines for protein production, the adherentG71 cells were pre-adapted to serum-free growth medium using a protocolfor adapting anchorage-dependent, serum-dependent mammalian cells tohigh density serum-free suspension culture (Sinacore et al., Mol.Biotechnol. 15(3):249-257, 2000), resulting in the serum-free suspensionculture adapted cell line, G71S. Alternatively, adherent G71 cells,after being stably transfected as described infra, may be adapted toserum-free growth medium as outlined in Sinacore et al.

Paired combinations of the human SUMF1 and human GALNS expressionvectors (Example I), either pcDNA4 SUMF1 plus pCIN4 4A, BMAR SUMF1 plusBMAR 4A, or PMAR SUMF1 plus PMAR 4A, were transfected following theMARtech II protocol as described by Selexis into G71S cells grown inculture medium supplemented with Antibiotic-Antimycotic Solution (100 IUPenicillin, 10 mg Streptomycin, 25 μg Amphotericin B, Cellgro).Transfectant pools were grown in UltraCHO medium (Cambrex) supplementedwith 5% γ-irradiated fetal bovine serum (FBS, JRH), 200 μg/mL G418 (AGScientific) and 200 μg/mL Zeocin (Invitrogen), and cloned by limitingdilution in 96-well plates in the same growth medium. Clone growth wasmonitored by Cell Screen (Innovatis) imaging. All clones were screenedusing an enzyme capture activity ELISA for active GALNS (see ExampleIV). Cellular productivity was calculated by dividing enzyme captureactivity ELISA for GALNS activity by cell growth (Vi-Cell, BeckmanCoulter) per day, over a period of 4 days.

202 G71S clones were generated and screened for active GALNS: 86 clonesco-transfected with pcDNA4 SUMF1 plus pCIN 4A, 65 clones co-transfectedwith BMAR SUMF1 plus BMAR 4A, and 51 clones co-transfected with PMARSUMF1 plus PMAR 4A. Clones were initially selected on the basis of highlevels of active GALNS from the 96-well tissue culture plates (FIG. 6A).GALNS activity was measured using an enzyme capture activity ELISA andrepresented in ng/mL (y-axis). The x-axis shows the threeco-transfection conditions used for SUMF1 and GALNS expression: hCMVpromoter without MAR, hCMV promoter with MAR, and SV40 promoter withMAR. Each bar represents a single clone from the respective population.Cell density was not accounted for in this 96-well clone screen and notall of the co-transfected G71S clones are displayed in this figure.

The highest active GALNS producing G71S clones were chosen forproductivity analysis (FIG. 6B). Daily cellular productivity wasmeasured in pg/cell/day and obtained by dividing the GALNS activity bythe cell density for that day. This figure displays the fourth day (96hours) after seeding at 5×10⁵ cells/flask. The clones were assayed forGALNS using an enzyme capture activity ELISA in pg/cell/day (y-axis).Positive controls consisted of GALNS expressing BHK and CHO clones(BioMarin). Each vertical bar represents a single clone. Active GALNSwas produced by pCIN 4A clones, but only marginally above the backgroundof the assay.

Analysis of clones by the 96-well screen and 4-day productivity assaydemonstrated that co-transfection of expression vectors with MARelements increased the productivity of G71S clones as compared toco-transfection of expression vectors without MAR elements. The BMAR4A+BMAR SUMF1 co-transfected clones demonstrated fast pool generation,rapid clone growth, and ability to produce greater than 2-fold moreactive GALNS than the highest producing PMAR 4A clones, and up to a10-fold increase over CHO 4A and BHK 4A clones lacking MAR elements.

The GALNS expressing G71S clones were adapted to serum-free growthmedium using the protocol outlined in Sinacore et al., Mol. Biotechnol.15(3):249-257, 2000. The entire adaptation was done in the presence ofboth selection agents (zeocin at 200 μg/mL and neomycin at 200 μg/mL).The GALNS expressing G71 clones cultured in T-flasks were split asfollows: (1) into a 125 mL shaker with the Cambrex UltraCHO medium and5% FBS (lot #8L2242); (2) into a 125 mL shaker with the JRH 302M medium(production medium) and 5% FBS; and (3) into T-flasks as a back-up(UltraCHO, 5% FBS). Once suspension cultures were established, adherentcells were discarded, and weaning from FBS was initiated. When thegrowth rate returned to >0.5 (1/day) for 3 passages and the viabilitywas >95%, the FBS concentration was reduced by 50%. The cells were leftat any given FBS concentration for a minimum of 3 passages. Once adaptedto growth in 2.5% FBS, the cells were taken directly into serum-freemedia. Cells were banked in fresh media with 10% (v/v) DMSO. A trialthaw was tested to insure that the cells survived the freeze process.Two GALNS expressing G71S clones from the BMAR 4A+BMAR SUMF1transfection, clones 4 and 5 took approximately 15 passages foradaptation to serum-free suspension culture. A GALNS expressing clonefrom the pcDNA4 SUMF1 plus pCIN 4A transfection, C6, was also isolatedand adapted to serum-free culture.

Paired combinations of the human SUMF1 and human GALNS expressionvectors (Example I), pcDNA4 SUMF1 plus pcDNA4-4A, were transfected intoG71S cells basically as described above, except 200 μg/mL Zeocin(Invitrogen) was used for selection. Six GALNS expressing clones, C2,C5, C7, C10, C11 and C30, were isolated and adapted to serum-freesuspension culture basically as described above.

Example III Large-Scale Culture of G71S Cell Lines Expressing HumanN-Acetylgalactosamine-6-Sulfatase (GALNS)

The objective was to measure enzyme production from the G71S clonesexpressing human N-acetylgalactosamine-6-sulfatase (GALNS). Serum-freesuspension culture adapted G71S cell lines co-expressing human SUMF1 andhuman GALNS were cultured in large-scale and assessed for active GALNSenzyme production.

Since adaptation to serum-free suspension culture was relatively quickfor the G71S host cell line, it was decided that production could bedone in a WAVE bioreactor operated in perfusion mode. The WAVEbioreactor allows greater flexibility in inoculum volume becausescale-up can be done directly in the bag, reducing the risk ofcontamination and expediting the production of material. FIG. 7 showsthe schematic of WAVE bioreactor setup. The diagram shows, in perfusionmode, that a load cell monitors the media volume in the bag bydetermining the weight of the bag and adjusting the feed and harvestrates to maintain the desired volume. In the 10 L bag, the pH is alsocontrolled to the desired set-point by a probe that is inserted into thebag.

The material from the GALNS expressing G71S clones 4 and 5 was producedat the 1 L scale. The culture pH was not controlled in these runs. Theoperational limitation of the WAVE bag is a throughput of 3 vesselvolumes a day (VV/day). In order to prevent any inactivation ofmaterial, the target cell specific perfusion rate (CSPR) was 0.3nl/cell/day, resulting in an average residence time of eight hours forthe GALNS enzymes. Therefore, the cell density in the bag was maintainedat approximately 10-12×10⁶ cells/mL. The growth rate for GALNSexpressing G71S clones 4 and 5 was 0.16 and 0.20, respectively. Bleedsto maintain target cell density were done directly from the bag.

The harvest fluid pH was adjusted to a pH between 5.5 and 6.5 tomaintain enzymatic activity, since GALNS had previously been shown to bestable at pH 6. This was accomplished by a timed bolus addition of 5% byvolume pH 4.0 sodium citrate buffer mixed in line with harvest comingoff the reactor. The adjusted harvest fluid was stored at 4° C. prior todownstream processing. The two GALNS expressing G71S clones 4 and 5averaged titers of about 4.2 mg/L with an associated specificproductivity of about 1.25 pg/cell/day.

The GALNS expressing G71S clones, C2, C5, C6, C7, C10, C11 and C30, weresimilarly cultured in large-scale and assessed for active GALNS enzymeproduction.

Example IV Measurement of the Concentration and Activity of HumanN-Acetylgalactosamine-6-Sulfatase (GALNS)

Enzyme linked immunosorbant assays (ELISAs) were developed to measureGALNS enzyme concentration and activity from the G71S clonesco-expressing human SUMF1 and human N-acetylgalactosamine-6-sulfatase(GALNS).

Enzyme Capture Activity ELISA

The enzyme capture activity ELISA measures the activity of GALNS enzymein solid phase, following the capture by an anti-GALNS specific antibodybound to an ELISA plate.

Buffers. Buffer A (Carbonate Buffer): dissolve 3.09 grams of Na₂CO₃ and5.88 grams of NaHCO₃ in 900 mL of de-ionized (DI) H₂O, then add DI H₂Oto a final volume of 1000 mL. Check that the pH is between 9.4 and 9.6,then filter-sterilize. To completely coat one 96-well microplate with100 μL per well, dilute 19 μL of an anti-GALNS antibody into one tube(12 mL). Buffer B (ELISA Blocking Buffer and Serial Dilution Buffer): 1×Acidic PBS, 0.05% Tween-20 and 2% BSA, adjusted to pH 6.5 with aceticacid. Buffer B^(W) (Wash Buffer): 100 mM NaOAc and 0.05% Tween-20,adjusted to pH 6.5 with acetic acid. Buffer C (Substrate Buffer): 25 mMSodium Acetate, 1 mM NaCl, 0.5 mg/mL desalted BSA and 0.01% sodiumazide, adjusted to pH 4.0 with glacial acetic acid. Buffer D(β-Galactosidase Buffer): 300 mM sodium phosphate dibasic, 0.1 mg/mlBSA, 0.01% sodium azide and 0.01% Tween-20, adjusted to pH 7.2 withphosphoric acid. Buffer E (Stop Buffer): 350 mM glycine and 440 mMcarbonate buffer, adjusted to pH 10.7 with 6 M NaOH.

Reagents. Anti-GALNS IgG antibody: polyclonal rabbit antibodies areProtein G purified from serum. In D-PBS, total protein=3.17 mg/mL (BCA).Aliquots (19 μL) are stored at −20° C. for one-time use each.4MU-Gal-6-S Substrate (Solid; 440 MW): 100 mM stock prepared in DI waterand stored at 4° C. β-Galactosidase (Sigma G-4155): dilute to 12 μg/mLin Buffer D prior to use.

Protocol: Bind anti-GALNS antibody to plate: a Nunc MaxiSorp ELISA plate(Nalge/Nunc International, Fisher #12-565-135) is coated with anti-GALNSantibody at a final protein concentration of 5 μg/mL in Buffer A. Toprepare this solution, thaw one 19 μL aliquot, spin briefly (10 sec) ina microcentrifuge to collect the liquid. Transfer all 19 μL into 12 mLof Buffer A. Mix vigorously by inversion, then pour into a reservoir,followed by plate loading (100 μL per well) using a multi-channelpipettor. Cover the plate and incubate at 4° C. overnight. Removeunbound anti-GALNS antibody: wash the plate by flooding with BufferB^(W) three times. Block: block the plate with Buffer B (320 μL perwell), then cover the plate and incubate at 37° C. for 1 hr. Prepare adilution series of purified GALNS standard and test samples (unknowns)during the block step: the standard is diluted in Buffer B to the highend of the linear range of the assay (128 ng/mL in Row A), then seriallydiluted (2-fold) in rows B-G on a 96-well plate. Lane H is buffer blank(i.e., no GALNS enzyme). First, prepare 500 μL of a concentration at 128ng/mL in Buffer B. Then, dilute serial 2-fold in the Buffer B (250 μLinto 250 μL) until reaching 2 ng/mL. Remove blocking buffer: after theblock step, Buffer B is discarded. Bind GALNS enzyme standard and testsamples to anti-GALNS antibody: load the plate with 100 μL/well of theserially diluted standard and test samples (run in duplicate). Cover theplate and incubate at 37° C. for 1 hr. Remove GALNS inhibitors: wash theplate by flooding with Buffer B^(W), three times. Add GALNS substrate(first reaction): prepare enough final substrate solution for loading100 μL per well (prepared no more than 1 hour before use). Dilute the4MU-Gal-6-S stock solution (100 mM) to 1 mM in Buffer C. Load 100 μL perwell. Cover the plate and incubate at 37° C. for 30 min. Addβ-Galactosidase(second reaction): add 50 μL of 12 μg/ml β-galactosidasein Buffer D to each well. Cover the plate and incubate at 37° C. for 15min. Stop reaction: add 100 μL of Buffer E (stop buffer) to each well toionize released 4MU. Transfer to fluoroplate: transfer (8 wells at atime) 200 μL of the 250 μL from each well of the ELISA plate to a blackuntreated flat-bottom microtiter plate (Fluoroplate, Costar #3915). Readfluorescence: read the plate in a Gemini plate reader (Molecular DevicesCorporation) using the SOFTmax PRO program (366 nm excitation, 446 nmemission, 435 nm cutoff).

GALNS Elisa

The GALNS ELISA measures the concentration of the GALNS enzyme in cellculture conditioned medium or other process samples using a sandwichimmunoassay.

Buffers. Buffer A (Carbonate Buffer): dissolve 3.09 grams of Na₂CO₃ and5.88 grams of NaHCO₃ in 900 mL of de-ionized (DI) H₂O, then add DI H₂Oto a final volume of 1000 mL. Check that the pH is between 9.4 and 9.6,then filter-sterilize. To completely coat one 96-well microplate with100 μL per well, dilute 19 μL of anti-GALNS antibody into one tube (12mL). Buffer B (ELISA Blocking Buffer and Serial Dilution Buffer): 1×acidic PBS, 0.05% Tween-20 and 2% BSA, adjusted to pH 6.5 with aceticacid. Buffer B^(W) (Wash Buffer): 100 mM NaOAc and 0.05% Tween-20,adjusted to pH 6.5 with acetic acid. Buffer F (Stop Buffer): 2N H₂O₄: in600 mL total, add 100 mL of 12N H₂SO₄ and 500 mL MilliQ water.

Reagents. Anti-GALNS IgG antibody: rabbit polyclonal antibodies areProtein G purified from serum. In D-PBS, total protein=3.17 mg/mL (BCA).Aliquots (19 μL) are stored at −20° C. for one-time use each.HRP-conjugated detecting antibody (RIVAH): the final conjugated antibodyis diluted 1:100 into D-PBS/1% BSA and stored in 120 μL aliquots at −20°C. for one-time use. TMB EIA Substrate Kit (BioRad #172-1067).

Protocol. Bind anti-GALNS antibody to the plate: a Nunc MaxiSorp ELISAplate (Nalge/Nunc International, Fisher #12-565-135) is coated withanti-GALNS antibody at a final protein concentration of 5 μg/mL inBuffer A. To prepare this solution, thaw one 19 μL aliquot, spin briefly(10 sec) in a microcentrifuge to collect the liquid. Transfer all 19 μLinto 12 mL of Buffer A. Mix vigorously by inversion, then pour into areservoir, followed by plate loading (100 μL per well) using amulti-channel pipettor. Cover the plate and incubate at 37° C.(convection incubator) for 2 hr. Do not use a hot block. Remove unboundanti-GALNS antibody: wash the plate by flooding with Buffer B^(W), threetimes. Block: block the plate with Buffer B (320 μL per well), thencover the plate and incubate at 37° C. for 1 hr. Prepare dilution seriesof purified GALNS standard and test samples (unknowns) during blockstep: the standard is diluted in Buffer B to the high end of the linearrange of the assay (40 ng/mL in Row A), then serially diluted (2-fold)in rows B-G on a 96-well plate. Lane H is buffer blank (i.e., no GALNSenzyme). First, prepare 500 μL of a concentration at 40 ng/mL in BufferB. Then, dilute serial 2-fold in the Buffer B (250 μL into 250 μL) untilreaching 0.625 ng/mL. Remove blocking buffer: after the block step,Buffer B is discarded. Bind GALNS enzyme standard and test samples toanti-GALNS antibody: load the plate with 100 μL/well of the seriallydiluted standard and test samples (run in duplicate). Cover the plateand incubate at 37° C. for 1 hr. Wash: wash the plate by flooding withBuffer B^(W), three times. Bind detecting antibody conjugate: thaw onealiquot (120 μL) of antibody RIVAH, spin briefly (10 sec) in amicrocentrifuge to collect the liquid. Dilute all 120 μL into 11.9 mLBuffer B and vigorously invert the tube to mix. Pour into reservoir andadd 100 μL per well with the multichannel pipettor. Cover the plate andincubate at 37° C. for 30 min. Wash: wash the plate by flooding withBuffer B^(W), three times. TMB substrate: prepare the final substratesolution by mixing 1.2 mL of Solution B with 10.8 mL of Solution A. Pourinto reservoir and add 100 μL per well with the multichannel pipettor.Cover the plate and incubate at 37° C. for 15 min. Stop solution:Pipette 12 mL of 2N H₂SO₄ stop solution into reservoir and add 100 μLper well with the multichannel pipettor. Tap gently to mix. Read A450:read plate in the plate reader.

GALNS Specific Activity Assay

The GALNS specific activity assay measures the enzymatic activity ofGALNS in solution using a GALNS-specific substrate.

Buffers. MilliQ H₂O is used for all buffers. Dilution Buffer (DB): for 1L of DB, dissolve 1.74 mL acetic acid, 0.75 g sodium acetate, 233.6 mgNaCl, 2 mL of 50% Tween-20 and 10 mL of 1% sodium azide into MilliQ H₂O,and adjust the pH to 4.0+/−0.5 with 0.1 M NaOH if the pH is less than3.95 and with 0.1 M acetic acid if the pH is greater than 4.05. Thefinal concentrations are: 19.5 mM acetic acid, 5.5 mM sodium acetate, 1mM NaCl, 0.1% Tween-20 and 0.01% sodium azide. Phosphate Buffer (PB):for 1L PB, dissolve 13.9 g NaH₂PO₄—H₂O and 55 g NaHPO₄-7H₂O in MilliQH₂O, and adjust the pH to 7.2. The final concentration is 300 mM NaPi.Stop Buffer (SB): for 1 L SB, dissolve 26.2 g glycine and 46.6 g sodiumcarbonate in MilliQ H₂O, and adjust the pH to 10.6 with NaOH. AssayBuffer (AB): dilute 4MU-Gal-65 stock 1:50 in DB (2 mM final).β-Galactosidase Buffer (βGB): 25 μg/mL β-Galactosidase in 300 mM NaPi,pH 7.2.

Reagents. 4MU-Gal-65: 100 mM in H₂O (Toronto Research Chemicals Cat.#M334480). β-Galactosidase: Sigma G-4155. 4-methylumbelliferone (4MUstandard): Sigma M-1381 (10 mM stock in DMSO).

Protocol. Perform serial dilutions of the GALNS enzyme. For purified andformulated GALNS (˜1.5 mg/ml), dilute samples 1:10,000 in low proteinadhesion microcentrifuge tubes (USA Scientific Cat#1415-2600) containingDB, prior to 1:1 serial dilutions. Place 100 μL of DB in a lowprotein-binding 96-well plate. In the first row, pipette 100 μL of GALNSsample. Now serially dilute (1:1) down the plate (A-G on 96-wellplates). No sample is added to well H (blank) The linear range of thisassay is 1-75 ng/mL. Use the same procedure for preparing the 4MUstandard curve. Dilute 10 mM 4MU stock in DMSO 1:100 in DB. Start 4MUstandard curve by adding 50 μL of 50 μM 4MU in the first well, thenserially dilute. Add 50 μL of the substrate diluted in AB (2 mM4MU-Galactose-6S in DB) to a 96-well fluorescent plate. Pre-incubatesubstrate for 10 min at 37° C. Add 50 μL of the 100 μL serial dilutionsof GALNS and 4MU standards to the 50 μL of substrate in AB. Incubate at37° C. for 30 min (this first reaction removes the sulfate from thesubstrate), quench the first reaction and start the second reaction byadding 50 μL of β-Galactosidase (dilute β-galactosidase stock to 25μg/mL in βGB. Phosphate inhibits GALNS and the increase in pH also stopsthe GALNS reaction. The resulting pH is now in the optimum pH range ofβ-galactosidases. Incubate this second reaction for 15 min at 37° C.Ionize released 4MU by adding 100 μL of SB. Read Ex355 Em460 on 96-wellfluorescent plate reader. Enzyme activity calculations (at 37° C. in pH4.0 buffer): 1 unit=μmol 4MU released/min; activity=μmol 4MU/min/mL;specific activity=μmol 4MU/min/mg. Protein concentration calculation:use extinction coefficient of GALNS (1 mg/mL=1.708 Absorbance Units at280 nm).

Example V Purification of Human N-Acetylgalactosamine-6-Sulfatase(GALNS)

The objective was to obtain a large quantity of recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS). Stably transfected G71 cellsco-expressing human SUMF1 and human GALNS were grown under bioreactorculture conditions, and active GALNS enzyme was purified from the cellmedium.

Liquid Chromatography Apparatus. Amersham Pharmacia Biotech AKTAexplorer 900 system, utilizing Unicorn control software.

Protein Analytical Methods. Standard procedures were followed forSDS-PAGE, Coomassie Blue staining (B101-02-COOM), Western blotting andBradford protein assays. The purification runs were assessed by yield ofactivity, and the purity of the GALNS product was assessed visually bySDS-PAGE. The presence of processed impurities was detected by Westernblotting using an anti-GALNS antibody. Protein concentration wasmeasured using a Bradford protein assay. The concentration of the finalpurified GALNS protein was measured by A₂₈₀ measurement using anextinction coefficient of 1.708.

Chromatography Resins. Blue Sepharose 6 FF (GE Healthcare, lot #306346)and Fractogel SE Hi-Cap (Merck KgaA, FC040894449).

GALNS Enzyme Activity Determinations. The GALNS specific activity wasdetermined using a small fluorescent substrate4-methylumbelliferyl-6-S-GAL (4-MU-6-S-GAL). The GALNS specific activityassay involves a two-step reaction, wherein addition of β-galactosidaseis necessary after incubation of GALNS with the substrate for a certaintime to release the fluorescent tag. Measurements are made using afluorescence plate reader.

A 10DG desalting column (Bio-RAD) was equilibrated with equilibrationbuffer (EQB, 50 mM NaOAc, 10 mM NaCl, pH 5.8). MilliQ H₂O was used forall buffers. Three (3) mL of purified GALNS (0.5-2 mg/mL) was loadedonto the desalting column, eluted and collected in 4 mL aliquots inseparate test tubes using EQB. The protein concentration was calculatedusing the extinction coefficient of GALNS (1 mg/mL=1.708 AbsorbanceUnits at 280 nm).

Desalted GALNS samples were serially diluted (1:1) in dilution buffer(DB, 50 mM NaOAc, 1 mM NaCl, pH 4.0+0.5 mg/mL BSA). The BSA stock wasdesalted before using by loading 50 mg/mL BSA stock (no more than 5% CV)onto a G25 column previously equilibrated with milliQ H₂O. 100 μL of thedesalted GALNS sample was pipetted in the first row of a low proteinbinding 96-well plate, and the serially diluted GALNS samples werepipetted down the plate (rows A-G on 96-well plates). 100 μL of DB waspipetted into the last well (H). The top end of the linear range of thisassay is 200 ng/mL, and the linear range is 3-200 ng/mL. The sameprocedure was performed for preparing the standard curve with4-methylumbelliferone (4MU) (Sigma M-1381, 10 mM stock in DMSO). 50 μLof the 100 μL serial dilutions of GALNS and 4MU were transferred to anew 96-well fluorescent plate (black bottom plate). 50 μL of 2 mM4MU-Galactose-6S (in milliQ H₂O) was added to the samples to be assayed,and incubated at 37° C. for 30 minutes. This first reaction wasquenched, and a second reaction was initiated by adding 50 μL ofβ-Galactosidase (Sigma G-4155, stock diluted to 12 μg/mL in 300 mM NaPi,pH 7.2), and incubated at 37° C. for 15 minutes. Released 4MU wasionized by adding 100 μL of stop buffer (Glycine/Carbonate, pH 10.6).The plates were read on 96-well fluorescent plate reader (excitation 355nm, emission 460 nm). 1 Unit is defined as 1 μmol 4MU released/min,enzyme activity is given in μmol 4MU/min/mL, and specific activity isgiven in μmol 4MU/min/mg, all at 37° C. in pH 4.0 buffer.

First Purification Process. A first purification process included anultrafiltration (UF) step followed by a 2-column purification process.

1. Harvest Filtration (HF): the bioreactor material was 0.2 μm sterilefiltered.

2. Ultrafiltration (UF): the bioreactor material was concentrated 10-20×by ultrafiltration through a 30 kD Sartocon membrane.

3. pH 4.5 Adjust: the concentrated bioreactor material (UF (20×)) wasadjusted to pH 4.5 with pH adjust buffer (1.75 M NaOAc, pH 4.0) at roomtemperature and sterile filtered before loading on a Blue Sepharosecolumn.

4. Blue Sepharose 6 Fast Flow (FF): the pH 4.5 adjusted UF (20×) wasloaded onto a Blue Sepharose column and the GALNS protein was eluted asshown in Table 1 and FIG. 9A.

TABLE 1 Blue Sepharose 6 Fast Flow Chromatography Step CV* BufferEquilibration 5 20 mM acetate/phosphate, 50 mM NaCl, pH 4.5 Load UFproduct, adjusted to pH4.5, filtered Wash 1 4 20 mM acetate/phosphate,50 mM NaCl, pH 4.5 Wash 2 8 20 mM acetate/phosphate, 50 mM NaCl, pH 6.0Elution 8 20 mM acetate/phosphate, 100 mM NaCl, pH 7.0 Strip 5 20 mMacetate/phosphate, 1 M NaCl, pH 7.0 Sanitization 4 0.1 N NaOH, 0.5 hourRegeneration 5 H₂O Storage 3 20% ETOH *CV: column volumes. Flow rate =92 cm hr⁻¹

5. Fractogel SE Hi-Cap: the eluate from the Blue Sepharose column wasadjusted to pH 4.3 and loaded onto a Fractogel SE Hi-Cap column and theGALNS protein was eluted as shown in Table 2 and FIG. 9B.

TABLE 2 Fractogel SE Hi-Cap Chromatography Step CV* Buffer Equilibration5 20 mM acetate/phosphate, 50 mM NaCl, pH 4.3 Load Blue Sepharose Eluateadjusted to pH 4.3 and diluted 1:1 with MQ water Wash 1 5 20 mMacetate/phosphate, 50 mM NaCl, pH 5.0 Wash 2 5 20 mM acetate/phosphate,50 mM NaCl, pH 5.5 Elution 20 20 mM acetate/phosphate, 50-350 mM NaClgradient, pH 5.5 Regeneration 1 5 20 mM acetate/phosphate, 500 mM NaCl,pH 5.5 Regeneration 2 5 20 mM acetate/phosphate, 50 mM NaCl, pH 4.3Sanitization 5 0.5 N NaOH, 0.5 hour Regeneration 3 4 H₂O Storage 3 20%EtOH *CV: column volumes. Flow rate = 150 cm hr⁻¹

The GALNS protein in the eluate was collected by fractionation,discarding the pre-elution shoulder and post-elution tail.

6. Final UF/HF: the eluate from the Fractogel SE Hi-CAP column wasconcentrated by ultrafiltration and sterile filtered as described above.

Formulation. The purified GALNS protein was formulated in 10 mM NaOAc, 1mM NaH₂PO₄, 0.005% Tween-80, pH 5.5.

Stability Studies. Stability of the final formulated purified GALNS wasmonitored at 4° C. and −70° C. as a function of time by storing smallaliquots of the GALNS samples at the respective temperatures. At certaintime points, aliquots of frozen samples were quickly thawed in a 37° C.waterbath before activity measurements. FIG. 8 shows that the purifiedGALNS was stable at 4° C. and −70° C. over a period of up to at least 79days in the formulation buffer.

First Purification Process Results. Table 3 shows the purificationyields for three preparations of GALNS protein produced from G71S clone4 in a suspension culture bioreactor. Purity was estimated visually bySDS-PAGE to be about 95% in all cases.

TABLE 3 Human N-Acetylgalactosamine-6-Sulfatase (GALNS) PurificationYields from G71S Clone 4 from WAVE Reactor Yield Steps Prep 1 Prep 2Prep 3 Average Std Dev UF N/A 100 100 100 0 Blue Sepharose 6 FF 93 103101 99 5.3 SE Hi-Cap 90 87 90 89 1.7

FIG. 9 shows an SDS-PAGE of the GALNS protein separated by (A) BlueSepharose 6 Fast Flow chromatography followed by (B) Fractogel SE Hi-CAPchromatography. The gels were stained with Coomassie Blue (left) oranti-GALNS antibody (right). For the Western blots, the anti-GALNSrabbit antibody was diluted to 1:5000, and the secondary antibody was ananti-alkaline phosphatase rabbit antibody. The GALNS protein has anapparent molecular weight of ˜55-60 kDa on SDS-PAGE, consistent withexpected size of the secreted pre-processed (precursor) form of theenzyme lacking the 26 amino acid residue signal peptide, and alsolacking the cleavage after position 325.

N-Terminus Characterization. The N-terminus of the purified GALNSprotein was determined by LC/MS. The N-terminal sequence was APQPPN,which corresponds to the predicted N-terminus of the secreted form ofGALNS lacking the 26 amino acid residue signal peptide (compare thehuman GALNS polypeptide sequences in FIG. 4 and FIG. 5).

Second Purification Process. A second purification process included anultrafiltration/diafiltration (UF/DF) step followed by a 3-columnpurification process.

1. Ultrafiltration (UF/DF): the bioreactor material was concentrated 20×by ultrafiltration/diafiltration through a 30 kD Sartocon membrane at pH5.5.

2. pH 4.5 Adjust: the concentrated bioreactor material (UF/DF (20×)) wasadjusted to pH 4.5 with pH adjust buffer (1.75 M NaOAc, pH 4.0) at roomtemperature and sterile filtered before loading on a Fractogel EMD SEHi-Cap column.

3. Fractogel EMD SE Hi-Cap: the pH 4.5 adjusted UF/DF (20×) was loadedonto a Fractogel EMD SE Hi-Cap column, washed sequentially with 10 mMacetate/phosphate, 50 mM NaCl, pH 4.5 and 10 mM acetate/phosphate, 50 mMNaCl, pH 5.0, and the GALNS protein was eluted with 10 mMacetate/phosphate, 140 mM NaCl, pH 5.0.

5. Zn-chelating Sepharose FF: the eluate from the Fractogel EMD SEHi-Cap column was adjusted to 500 mM NaCl, pH 7.0 and loaded onto aZn-chelating Sepharose FF (Zn-IMAC) column, washed with 10 mMacetate/phosphate, 125 mM NaCl, 10 mM imidazole, pH 7.0, and the GALNSprotein was eluted with 10 mM acetate/phosphate, 125 mM NaCl, 90 mMimidazole, pH 7.0.

6. pH 3.5 Adjust: the eluate from the Zn-chelating Sepharose FF columncontaining the GALNS protein was adjusted to pH 3.5 for low pH viralinactivation and then adjusted to 10 mM acetate/phosphate, 2 M NaCl, pH5.0.

7. ToyoPearl Butyl 650M: the low pH adjusted eluate from theZn-chelating Sepharose FF column, was loaded onto a ToyoPearl Butyl 650Mcolumn, washed with 10 mM acetate/phosphate, 2 M NaCl, pH 5.0., and theGALNS protein was eluted with 10 mM acetate/phosphate, 0.7 M NaCl, pH5.0.

8. Final UF/HF: the eluate from the ToyoPearl Butyl 650M eluate wasultra-filtered and dia-filtered in 20 mM acetate, 1 mM phosphate, 150 mMNaCl, pH 5.5.

Formulation. The purified GALNS protein was formulated in 10 mMNaOAc/HOAc, 1 mM NaH₂PO₄, 150 mM NaCl, 0.01% Tween-20, pH 5.5.

Second Purification Process Results. Table 4 shows the recovery forGALNS protein produced from G71S clone C2 in a suspension culturebioreactor using the second purification process. Purity of theformulated GALNS enzyme (i.e., precursor and mature or processed formstogether) was about 98% as determined by C3 RP-HPLC. The percentage ofthe precursor form of the GALNS enzyme was about 85% as determined bySDS-capillary gel electrophoresis.

TABLE 4 Human N-Acetylgalactosamine-6-Sulfatase (GALNS) Recovery forG71S Clone C2 Process Step Recovery (%) pH Adjust 96 Fractogel SE Hi-CapColumn 98 Zn-IMAC Column 89 Low pH Viral Inactivation 89 ToyoPearl Butyl650M Column 99 Formulation 99 Overall 70

FIG. 10 shows an SDS-PAGE of the GALNS enzyme separated byultrafiltration/diafiltration (UF/DF), Fractogel SE Hi-CAPchromatography, Zn-chelating Sepharose FF chromatography and ToyoPearlButyl 650M chromatography. The gels were stained with Coomassie Blue(top left), anti-GALNS antibody (top right), anti-Cathepsin L (bottomleft) and anti-CHO proteins (CHOP, bottom right). For the Western blots,the anti-GALNS rabbit polyclonal antibody was diluted to 1:5000, and thesecondary antibody was an anti-rabbit AP conjugate; the anti-Cathepsin Lgoat polyclonal antibody was diluted to 1:1000, and the secondaryantibody was an anti-goat HRP conjugate; and the anti-CHOP rabbitpolyclonal antibody was diluted to 1:1000, and the secondary antibodywas an anti-rabbit HRP conjugate. The precursor GALNS enzyme has anapparent molecular weight of ˜55-60 kDa on SDS-PAGE, and the mature orprocessed forms of GALNS enzyme have apparent molecular weights of ˜39kDa and ˜19 kDa on SDS-PAGE.

Summary of First Purification Process. The GALNS enzyme was purifiedusing a purification train that had been modified from a standard train(see Table 5). Bioreactor harvest material was 0.2 μm sterile filteredand kept at 4° C. before loading onto the Blue-Sepharose capture column.The filtered bioreactor material was either loaded directly orconcentrated up to 15× by ultrafiltration. Modification of thepurification train was necessary because the downstream purificationsteps, SP Sepharose chromatography followed by Phenyl Sepharosechromatography, did not yield sufficiently pure GALNS. Using SE Hi-Capchromatography as a replacement for the two downstream purificationcolumns resulted in a 2-column purification process, with the purity offinal material significantly improved, and the overall GALNS recoveryincreased significantly from to ˜22% to ˜80%. The purity of the GALNSenzyme (consisting essentially of the precursor form, see FIG. 9), asdetermined by C4-RP chromatography, was roughly estimated at >95%, andthe purified GALNS enzyme remained stable in formulation buffer for morethan 79 days at both 4° C. and at −70° C.

TABLE 5 First Human N-Acetylgalactosamine-6-Sulfatase (GALNS)Purification Train Step Normal Process Modified Process 1 HF (1X) HF(1X)  2* UF (5X) UF (15X) 3 pH 4.5 Adjust pH 4.5 Adjust 4 Blue-Sepharose6 FF Blue-Sepharose 6 FF 5 SP Sepharose SE Hi-Cap 6 Phenyl SepharoseHi-Sub Final UF/DF 7 Final UF/DF *This step is optional.

Summary of Second Purification Process. The GALNS enzyme was alsopurified using a second purification train (see Table 6). The overallGALNS recovery was about 70% and the purity of the GALNS enzyme(including both precursor and mature or processed forms, see FIG. 10),as determined by C4-RP chromatography, was roughly estimated to be about97%.

TABLE 6 Second Human N-Acetylgalactosamine-6-Sulfatase (GALNS)Purification Train Step Process 1 HF (1X) 2 UF/DF (20X) 3 pH 4.5 Adjust4 SE Hi-Cap 5 Zn-chelating Sepharose 6 pH 3.5 Adjust 7 ToyoPearl Butyl650M 8 Final UF/DF

These assays indicate that the protocols described above for preparingrecombinant lysosomal sulfatase enzymes provide an efficient method forproduction of large quantities of highly purified enzyme, in particularthe secreted pre-processed (precursor) form of humanN-acetylgalactosamine-6-sulfatase (GALNS).

Example VI Characterization of Purified HumanN-Acetylgalactosamine-6-Sulfatase (GALNS)

The G71 cell lines produce proteins (e.g., lysosomal enzymes) withgreater levels of high-mannose phosphorylation than is noted in anaverage mammalian cell line, and a correspondingly lower level ofunphosphorylated high-mannose oligosaccharides. A lysosomal sulfataseenzyme (e.g., recombinant human N-acetylgalactosamine-6-sulfatase(GALNS)), comprising a high level of bis-phosphorylated high-mannoseoligosaccharides, as defined herein, is compared to molecules obtainedin Canfield et al., U.S. Pat. No. 6,537,785, which do not comprisecomplex oligosaccharides, and exhibit only high mannoseoligosaccharides.

To determine levels of unphosphorylated high-mannose on a lysosomalsulfatase enzyme, one of skill in the art can use exoglycosidasesequencing of released oligosaccharides (“FACE sequencing”), to pinpointthe percentages of unphosphorylated high-mannose oligosaccharide chains.On a normal lot-release FACE profiling gel, unphosphorylated highmannose co-migrates with particular complex oligosaccharides (e.g.,oligomannose 6 and fully sialylated biantennary complex).Unphosphorylated high mannose is then differentiated from the otheroligosaccharides by enzymatic sequencing.

To determine if the purified lysosomal sulfatase enzyme (e.g.,recombinant human N-acetylgalactosamine-6-sulfatase (GALNS)) expressedin G71S cells exhibits increased phosphorylation, the level ofmannose-6-phosphate (M6P) on the lysosomal sulfatase enzyme wasdetermined, as well as the enzyme's ability to bind to the M6P receptor(MPR).

Recombinant human GALNS enzyme, expressed in G71S cells and purified,was analyzed by fluorescence assisted carbohydrate electrophoresis(FACE) and by chromatography on MPR-Sepharose resin. The FACE systemuses polyacrylamide gel electrophoresis to separate, quantify, anddetermine the sequence of oligosaccharides released from glycoproteins.The relative intensity of the oligomannose 7 bis-phosphate (O7P) band onFACE (Hague et al., Electrophoresis 19(15): 2612-20, 1998) and thepercent activity retained on the MPR column (Cacia et al., Biochemistry37(43): 15154-61, 1998) give reliable measures of phosphorylation levelper mole of protein.

Specific Activity. The specific activity of recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) was determined using a smallfluorescent substrate 4-methylumbelliferyl-6-S-GAL (4MU-Gal-6S) at 37°C. Using this assay, the specific activity of the purified GALNS was 165μmol/min/mg (0.165 U/mg).

Human Serum Stability. The ex vivo serum stability of the GALNS wasdetermined. Human serum (Sigma H-4522) was filter sterilized through a0.2 μm PES filter, and 4 mL of the filter sterilized human serum waspre-incubated in a T-25 cell culture flask for 1 hour at 37° C. in anatmosphere of 10% CO₂ (pH at this point is 7.4±0.1). 0.4 mL of desalted,purified GALNS (2 mg/mL purified GALNS was desalted into PBS usingBio-RAD 10DG columns) was added to the pre-incubated human serum, or aPBS control containing 0.5 mg/L BSA. 100 μL samples were withdrawn atdesignated time points (e.g., 0, 1, 3.5, 7.5 and 26 hours) and added to900 mL of quench buffer (QB, 50 mM NaOAc, pH 5.6+150 mM NaCl+0.5 mg/mLBSA+0.001% Tween-80). Samples were stored at 4° C. until ready formeasuring GALNS enzyme activity.

The GALNS enzyme activity was measured using the enzyme capture activityELISA. By extrapolating the exponential decay curve of % residual GALNSenzyme activity, the ex vivo serum half-life of the purified GALNS wasestimated to be 217 hours.

Uptake into Synoviocytes (Chrondrocytes). The ability of GALNS to betaken up into synoviocytes (chrondrocytes) was determined.

Chondrocytes (ATCC Number CRL-1832) are cultured in growth media (Ham'sF12+10% FBS) at 37° C. in 5% CO₂ in 12-well dishes. The analysis ofuptake of three samples requires 4×12 well plates. The purified GALNSsamples and a GALNS reference were diluted to 1 μM in acPBS/BSA (acidicPBS+200 μg/mL BSA). From the 1 μM stocks, uptake dilution curves forGALNS samples and reference were prepared: 50.5 μL (1 μM rhASB) into 5mL uptake assay diluent (UAD, DMEM+2 mM L-glutamine+0.5 mg/mL BSA),resulting in 10 nM GALNS samples and reference, which were furtherserially diluted to 5, 2.5, 1.25, 0.62, 0.31 and 0.16 nM by serialtwo-fold dilutions in UAD. The growth medium from the 12-well dishes ofconfluent chondrocytes was aspirated, 1 mL of either UAD (blank) orserial dilutions of GALNS samples or references were added to the wells,and incubated for 4 hours at 37° C. in a 10% CO₂ incubator. The uptakemedium was aspirated, tilting each dish for completeness, and each wellwas rinsed once with 1 mL PBS. The PBS was aspirated and thechondrocytes were detached by adding 0.5 mL trypsin/EDTA (0.25%Trypsin/0.1% EDTA (Mediatech 25-053-CI, lot 25053025)) per well. Afterdetachment from the plate, the chondrocytes were aliquoted intoprechilled-on-ice Eppendorf tubes (30 tubes total). The trypsinizedchondrocytes were cooled and then pelleted at low speed in a microfuge(4000 rpm for 3 minutes). The trypsin was aspirated completely, the cellpellet was rinsed with 1 mL PBS, repeating the microfuge and aspirationsteps once. 200 μL of cell lysis buffer (CLB, 50 mM sodium acetate, pH5.6+0.1% Triton X-100) was added to each tube. Cell pellets wereresuspended by pulse-vortexing three times. After resuspension, the celllysis mixtures were stored overnight at −80° C., or analyzed directly.

The cell lysates were thawed at room temperature and transferred to icewhen thawed. The cell lysates were vortexed to resuspend any visiblesolid material, and then spun in the microfuge at 14 Krpm for 10 minutesat 4° C. to pellet the insoluble material. The supernatants weretransferred to a fresh set of tubes and the pellet was discarded. ThenGALNS activity assay was performed on the supernatants. A seven-pointdilution curve (serial two-fold dilutions starting at 10 nM and endingat 0.16 nM) is usually performed which brackets the expected K_(uptake)fairly evenly on both sides. The molarity of the starting samples iscalculated by using the protein-only molecular weight.

The purified GALNS had a Kd for uptake into synoviocytes, based onsingle-site ligand binding, of 4.9 nM.

Mannose-6-Phosphate (M6P) Receptor Plate Binding Assay. The ability ofGALNS to bind to the mannose-6-phosphate (M6P) receptor was determinedin a plate binding assay. FluoroNunc high binding plates were coatedwith 4 μg/mL M6P receptor. The coated plates were washed twice with 250μL/well of wash buffer (WB, TBS+0.05% Tween 20) and nonspecific bindingwas blocked with 200 μL/well of blocking and dilution buffer (BDB,Pierce SuperBlock buffer, lot #CA46485). Plates were incubated for 1hour at room temperature (RT). During this block step, purified GALNSsamples (0.5-2 mg/mL stored at 4° C. for 2 weeks) were diluted to 10 nMin BDB, and then serially diluted in dilution buffer (DB, 50 mM NaOAc, 1mM NaCl, pH 4.0+0.5 mg/mL BSA) (250 μL+250 μL) down to 5, 2.5, 1.25,0.62, 0.31 and 0.16 nM. Blocked plates were washed with WB as above, anddiluted GALNS samples were dispensed into the wells in duplicate at 100μL/well and incubated 1 hour at RT. During this incubation step, 2 mMactivity substrate was prepared by diluting 0.1 mL of the 100 mM6S-galactose-4MU stock (stored in H₂O, −20° C.) into 5 mL DB, andprewarmed in a 37° C. water bath. After incubation, plates were washedtwice with WB as above, and 100 μL diluted substrate was added and theGALNS specific activity was determined

Using the assay, the purified GALNS had a Kd for binding to the M6Preceptor, based on single-site binding, of 2.4 nM.

Mannose-6-Phosphate (M6P) Receptor Column Binding. The ability of GALNSto bind to the mannose-6-phosphate (M6P) receptor was determined in acolumn binding assay. A M6P receptor column was prepared per themanufacturer's instructions. M6P receptor was from Peter Lobel'slaboratory, the column resin was NHS activated resin (Bio-RAD Affi-Gel15), and the column size was 0.7 mL. The M6P receptor column wasequilibrated with 10 column volumes (CV) of equilibration buffer (EQ,acidic PBS, pH 6.0 containing 5 mM β-glycerophosphate, 0.05% Tween 20, 5mM glucose-1-phosphate and 0.02% NaN₃) at a flow rate of 0.25 mL/min. 6μg of purified GALNS (per 200 μl) was loaded onto the M6P receptorcolumn at a flow rate of 0.1 mL/min. Unbound GALNS was washed off thecolumn with 10 CV of EQ at a flow rate of 0.25 mL/min. Bound GALNS waseluted off the column using a 0-100% elution buffer (EL, acidic PBS, pH6.0 containing 5 mM β-glycerophosphate, 0.05% Tween 20, 5 mMmannose-6-phosphate and 0.02% NaN₃) gradient (10 CV), followed by 2 CVof 100% EL. The column was re-equilibrated with 3 CV of EQ.

Using the GALNS ELISA, the percent of purified GALNS that bound to theM6P receptor was determined to be 56%.

Total Oligosaccharides Analysis by Capillary Electrophoresis (CE). Todetermine the level of mannose-6-phosphorylation on GALNS, the N-linkedcarbohydrate profile of the total oligosaccharides on the GALNS wasdetermined by capillary electrophoresis (CE) as described in Ma et al.,Anal. Chem. 71(22):5185-5192, 1999. The method used PNGase F to cleaveasparagine N-linked oligosaccharides. The cleaved oligosaccharides wereisolated and derivatized with fluorescent dye, and applied to a G10 spincolumn to remove excess dye. The purified, fluorescently labeledoligosaccharides were separated electrophoretically and peakssubsequently quantified using the MDQ-CE software (32 Karat Ver. 7.0).

Using this assay, the amounts of bis-phosphorylated mannose 7 (BPM7),mono-phosphorylated mannose 6 (MPM6) and sialic acid containingoligosaccharides for purified GALNS were 0.58 mol/mole enzyme, 0.08mol/mol enzyme and not detectable, respectively. The percent of GALNSproteins containing BPM7 was estimated to be 29%.

Bis7 Oligosaccharide Characterization. The location of thebis-phosphorylated mannose 7 (BPM7) oligosaccharides on the GALNS wasdetermined. The asparagine (Asn) residue at position 178 was N-linkedglycosylated to BPM7. The Asn residue at position 397 was not N-linkedglyosylated to BPM7, but was found to be predominantly oligomannose-typesugars.

Hydroxyapatite Affinity. An in vitro bone model was developed todetermine whether the GALNS had the ability to target to bone. A 4 mg/mLHTP-DNA grade hydroxyapatite (Bio-RAD) suspension was prepared andequilibrated in DBS+50 μg/mL BSA, pH 7.4. The purified GALNS, afteradding 50 μg/mL BSA, was desalted in DBS, pH 7.4. The desalted GALNS, ata final concentration of approximately 2 mg/mL, was serially diluted inDBS+50 μg/mL BSA, pH 7.4 in a 96-well plate. 50 μL of the seriallydiluted GALNS were transferred to 96-well filter plate (Millipore#MSGVN2210, hydrophilic PVDF, low protein binding, 22 μm pore size). 50μL of the hydroxyapatite suspension was added to the wells of the filterplate containing the serially diluted GALNS and incubated for 1 hour at37° C. with mild shaking. The plate was subjected to vacuum filtration.

The vacuum filter supernatants were analyzed by either HPLC or GALNSenzyme activity as described above. The purified GALNS had a Kd forhydroxyapatite of 3-4.0 μM.

The G71S cell line expressing human sulfatase modifying factor 1 (SUMF1)produces lysosomal sulfatase enzymes with higher amounts of activation(i.e., conversion of the active site cysteine residue toC_(α)-formylglycine (FGly)).

To determine if the purified recombinant lysosomal sulfatase enzyme(e.g., human N-acetylgalactosamine-6-sulfatase (GALNS)) co-expressedwith SUMF1 in G71S cells exhibits increased activation, the amount ofconversion of active site cysteine residue to FGly on the purifiedlysosomal sulfatase enzyme was determined.

GALNS Activation. The percent activation, i.e., percent conversion ofthe active site cysteine (Cys) cysteine residue C_(α)-formylglycine(FGly), of the GALNS was determined by LC/MS (TFA). The TIC/1000 forCys, FGly and Gly were 39, 1840 and 183, respectively, indicating thatabout 90% of the purified GALNS is in an active (i.e., FGly) form.

Summary. Table 7 shows a summary of the characterization of recombinantGALNS expressed in G71S clone 4 cells. Table 8 shows a summary of thecharacterization of recombinant GALNS expressed in G71S clone C2 cells.

TABLE 7 Characterization of Human N-Acetylgalactosamine-6-Sulfatase(GALNS) Produced from G71S Clone 4 Assay Category GALNS SpecificActivity: Activity/Antigen by ELISA 0.165 U/mg Specific Activity:Activity/Protein 7.7 U/mg Purity by C4-RP >95% (6 lots tested) Size bySEC 115 kDa (homodimer) Serum Stability at 37° C. 217 Hours Uptake:Chondrocytes 4.9 nM Uptake: Fibroblasts 5.0 nM Uptake: Osteoblasts 7.8nM Productivity 1.3 pg/cell/day Titer 4.2 mg/L M6P Receptor PlateBinding 2.4 nM M6P Receptor Column Binding: % Bound 56% M6P Content byCE: % of Total Carbohydrate 29% M6P Content: mol M6P/mol GALNS 0.58Sialic Acid Content be CE 1% Hydroxyapatite Affinity 4 μM Activation: %FGly 90%

TABLE 8 Characterization of Human N-Acetylgalactosamine-6-Sulfatase(GALNS) Produced from G71S Clone C2 Assay Category GALNS SpecificActivity: Activity/Protein 6.4 U/mg Purity by C4-RP 97% Size by SEC 115kDa (homodimer) Uptake: Fibroblasts 3.4 nM Titer 6.4 mg/L (4 lotstested) M6P Receptor Plate Binding 5.7 nM M6P Content by CE: % of TotalCarbohydrate 34.5% M6P Content: mol M6P/mol GALNS 0.69

These results demonstrate that the purified recombinant human GALNS hasa high level of activation, and high levels of mannose 6-phosphatephosphorylation. Thus, G71S cells co-expressing SUMF1 and a lysosomalsulfatase enzyme (i.e., GALNS) efficiently produce active highlyphosphorylated lysosomal sulfatase enzyme. The increased level of highmannose residues on such lysosomal sulfatase enzymes leads to increaseduptake by the MPR on cells.

Example VII Uptake and Activity of Recombinant HumanN-Acetylgalactosamine-6-Sulfatase (GALNS) in Morquio Chondrocytes InVitro

The uptake of recombinant human N-acetylgalactosamine-6-sulfatase(GALNS) by lysosomes of Morquio chondrocytes and the ability of GALNS todegrade keratan sulfate (KS) in vitro was evaluated.

Chondrocytes from patients with Mucopolysaccharidosis Type IVa (MPS IVa,Morquio Syndrome) have reduced GALNS activity and exhibit lysosomalaccumulation of KS. An in vitro model of MPS IVa was established usingchondrocytes isolated from iliac crest biopsies of a MPS IVa patient.Primary chondrocytes, however, de-differentiate and lose theirchondrocyte characteristics in culture. Thus, culture conditions wereestablished to induce chondrocyte differentiation in vitro.

Chondrocytes isolated from an MPS IVa patient, designated MQCH, werecultured in alginate beads in the presence of IGF-1, TGF-13,transferrin, insulin and ascorbic acid (Chondrocyte Growth Medium, Lonza#CC-3225). The culture medium was changed twice per week for theduration of the experiments, from 6 to 15 weeks. These cultureconditions induced expression of the chondrocyte phenotype anddifferentiation. These MQCH cells expressed chondrocyte markers,including sex determining region Y-box 9 (Sox 9), collagen II, collagenX, cartilage oligomeric matrix protein and aggregan mRNA, as measured byquantitative RT-PCR analysis using RNA isolated from cultures of MQCHcells. These cultured MQCH cells also elaborated extracellular matrix.

Confocal microscopy was performed to confirm that the MQCH cellsaccumulated KS. The MQCH cells in an 8-week culture were trypsinized,cytospun onto glass slides, fixed in acetone, and frozen until use.After thawing, the cells were rehydrated and stained using, as primaryand secondary antibodies, an anti-KS monoclonal antibody (Chemicon) andan Alexa-488 (green) conjugated goat anti-rabbit antibody, respectively.The MQ-CH cells displayed punctate intracellular staining, consistentwith lysosomal KS accumulation.

To determine whether purified recombinant human GALNS could be taken upby MQCH cells into lysosomes and degrade KS, a 6-week MQCH cell culturewas incubated with 10 nM recombinant human GALNS twice per week for 9weeks. GALNS uptake and KS clearance were measured by confocalmicroscopy. The primary antibodies used were: (a) an anti-GALNS rabbitpolyclonal antibody and an anti-Lysosomal Associated Membrane Protein-1(LAMP-1) monoclonal antibody, or (b) an anti-KS monoclonal antibody andan anti-LAMP-1 polyclonal antibody. The secondary antibodies used were:Alexa-488 (green) conjugated antibodies to detect anti-GALNS or anti-KSantibodies, or Alexa-555 or -594 (red) conjugated antibodies to detectanti-LAMP-1 antibodies. MQCH cell preparations were mounted in mountantcontaining DAPI, which stains nuclei.

Significant co-localization of the GALNS enzyme and KS with the lysosomemarker, LAMP-1, was observed in GALNS-treated MQCH cells. Upon exposureof MQCH to recombinant human GALNS, the amount of intracellular KS wasdecreased.

GALNS uptake was also measured using a GALNS enzyme capture ELISA and aGALNS specific activity ELISA, both described in Example IV above.Normal human chondrocytes (NHKC), which express GALNS, were used as apositive control. As shown in Tables 9 and 10, untreated MQCH cells hadno detectable GALNS enzyme or activity, whereas MCQH cells treated for 9weeks with 10 nM GALNS had significant GALNS enzyme and activity.

TABLE 9 GALNS Enzyme Capture ELISA Using MQCH Cells MQCH Cells NHKC Notreatment N.D.^(a) 0.12^(b) 10 nM GALNS for 9 wks 3.99 0.88 ^(a)Notdetected; ^(b)ng GALNS antigen/μg total protein

TABLE 10 GALNS Specific Activity Assay Using MQCH Cells MQCH Cells NHKCNo treatment N.D.^(a) 2.76^(b) 10 nM GALNS for 9 wks 3.68 5.15 ^(a)Notdetected; ^(b)GALNS activity/ng antigen

These results demonstrate that purified recombinant human GALNS is takenup by Morquio chondrocytes into lysosomes and can degrade lysosomal KSin vitro. These Morquio chondrocytes are useful as an in vitro efficacymodel to test lysosomal sulfatase enzymes, such as GALNS, which degradeKS.

Example VIII Activity of Recombinant Human Lysosomal Enzymes to DegradeNatural Substrates in a Cell-Based Assay In Vitro

Cell-based in vitro assays were developed to measure the activity ofrecombinant human lysosomal enzymes, e.g., lysosomal sulfatase enzymes,to degrade natural substrates.

The enzymatic activity of recombinant human lysosomal enzymes, e.g.,lysosomal sulfatase enzymes, is typically measured by a cell-free invitro assay using an artificial fluorogenic substrate (see Example 4 forGALNS). However, the enzyme activity measured is dependent on the sizeof the artificial substrate, i.e., number of monosaccharide units. Inaddition, the enzyme activity is measured in an environment that is notreflective of the situation in vivo. Thus, the cell-free in vitro assaydoes not take into account either the lysosomal enzyme's ability tocleave natural substrates, or its ability to be taken up into targetcells and localize to lysosomes.

A cell-based in vitro assay was developed to measure the activity of tworecombinant human lysosomal enzymes, alpha-L-iduronidase (IDU) andarylsulfatase B (ARSB), to degrade their natural substrates, i.e.,intracellular dermatan sulfate (DS)-containing substrates. DS containsvariably sulfated iduronic acid β(1-3) N-acetyl-galactosamine β(1-4)disaccharide units.

ARSB-deficient GM00519 human fibroblast cells or IDU-deficient GM01391human fibroblast cells were cultured to confluency in 12-well plates,and the cultures were maintained post-confluency for 3-6 weeks to allowfor accumulation of intracellular DS.

Post-confluent GM00519 or GM01391 cells were then exposed to saturatingdoses of recombinant human ARSB (10 nM) or recombinant human IDU (25nM), respectively, for 4-5 days. Untreated and lysosomal sulfataseenzyme-treated cells were harvested, lysed and centrifuged.

Lysosomal enzyme activity in the cell lysates was measured bydetermining the residual DS content of the cells by: (1) lysing thecells; (2) specifically digesting DS-containing substrates intodisaccharides using chondroitin ABC lyase (EC 4.2.2.4) in the celllysate; (3) labeling DS disaccharides with a fluorescent dye (e.g.,2-amino-acridone, AMAC); (4) separating the DS disaccharides (e.g., bycapillary zone electrophoresis, CZE); and (5) detecting the labeled DSdisaccharides (e.g., by laser-induced fluorescence, LIF). Such methodsare described, for example, in Zinellu et al., Electrophoresis2:2439-2447, 2007, and Lamari et al., J. Chromatogr. B 730:129-133, 1999(reviewed in Volpi et al., Electrophoresis 29:3095-3106, 2008).

Table 11 shows the percent degradation of DS using GM00519 cells treatedwith ARSB, as determined by measuring the amount of disaccharidecontaining N-acetylgalactosamine-4-sulfate (4S disaccharide), which isthe predominant DS disaccharide. Similar results were obtained usingGM01391 cells treated with IDU.

TABLE 11 Depletion of DS by ARSB in a Cell-Based In Vitro Assay Age ofCells GM00519 Cells (Weeks) (% Degradation)^(a) 3 86 4 92 5 92 6 89^(a)Percent degradation was calculated by measuring the area under thecurve of the 4S disaccharide detected in the CZE-LIF scan in lysatesfrom ARSB-treated as compared to untreated cells

The above assay indicated that target cells take up recombinant humanARSB and IDU, which are then localized to lysosomes, where they degradetheir natural substrate, intracellular DS.

A dose finding experiment was performed to determine the concentrationat which IDU becomes rate limiting in this cell-based assay. GM01391cells were cultured in 12-well plates. At 4 weeks post-confluency, thecells were exposed to various concentrations of IDU, from 0.8 nM to 25nM, for 6 or 26 hours. Cell lysates were prepared and processed asdescribed above. IDU was determined not to become rate limiting below 1nM.

In a second dose finding experiment, GM01391 cells at 3 weekspost-confluency were exposed to various concentrations of IDU, from 0.01to 0.2 nM, for 2 days. Cell lysates were prepared and processed asdescribed above. In this experiment, a known amount of an internalstandard monosaccharide, GlcNAc-6S, was spiked into the cell lysates tocontrol for recovery during processing. As shown in FIG. 11, a dosedependent decrease in the amount of DS substrate was observed in theIDU-treated GM01391 cells.

In a similar dose finding experiment, GM00519 cells at 3 weekspost-confluency were exposed to various concentrations of ARSB, from0.001 to 0.06 nM, for 5 days. Cell lysates were prepared and processedas described above. In this experiment, a known amount of an internalstandard monosaccharide, GlcNAc-6S, was spiked into the cell lysates tocontrol for recovery during processing. As shown in FIG. 12, a dosedependent decrease in the amount of DS substrate was observed in theARSB-treated GM00519 cells.

A cell-based in vitro assay was developed to measure the activity of arecombinant human lysosomal sulfatase enzyme, GALNS, to degrade itsnatural substrate, i.e., intracellular keratan sulfate (KS)-containingsubstrates.

GALNS-deficient MQCH cells were cultured as described in Example 7 aboveand treated with recombinant human GALNS at 1 or 10 nM. After treatment,MQCH cell lysates were prepared and digested with Keratanase II (EC3.2.1), which breaks down larger KS oligosaccharides into KSdisaccharides. The KS disaccharides were labeled with AMAC, separated byCZE and detected by LIF, as described above for DS disaccharides.GlcNAc-6S, a KS monosaccharide, was spiked into the cell lysates asinternal standard to control for recovery during processing. The amountsof two characteristic KS disaccharides, Gal6S-GlcNAc6S and Gal-GlcNAc6Swere measured, and the data obtained was corrected by the amount ofGlcNAc6S recovered. Table 12 shows the percent degradation of KS usingMQCH cells treated with GALNS, as determined by measuring the amount ofthe two characteristic KS disaccharides.

TABLE 12 Depletion of KS by GALNS in a Cell-Based In Vitro AssayGal6S-GlcNAc6S Gal-GlcNAc6S  1 nM GALNS 85.7^(a) 78.5^(b) 10 nM GALNS88.6 81.5 ^(a,b)Percent degradation was calculated by measuring the areaunder the curve of the Gal6S-GlcNAc6S and Gal-GlcNAc6S detected in theCZE-LIF scan in lysates from GALNS-treated as compared to untreated MQCHcells, and adjusting for the area under the curve of the spike controlGlcNAc6S

The above assay indicated that target cells take up recombinant humanGALNS, which is then localized to lysosomes, where GALNS degraded itsnatural substrate, intracellular KS.

Overall, these results demonstrated that the activity of recombinanthuman lysosomal enzymes, ARSB, IDU and GALNS, to degrade their naturalsubstrates can be measured and quantified in cell-based in vitro assays.It should be appreciated that this cell-based in vitro assay can bereadily modified to measure and quantify the activity of other lysosomalsulfatase enzymes, as well as a wide variety of recombinant lysosomalenzymes.

Example IX Delivery of Recombinant HumanN-Acetylgalactosamine-6-Sulfatase (GALNS) to Specific Tissues

The ability of recombinant human N-acetylgalactosamine-6-sulfatase(GALNS), expressed in G71 cells and purified, to be delivered tospecific tissues affected by, or associated with, deficiency of GALNSupon its administration into mice was evaluated.

The highly specific distribution of keratan sulfate gives the verycharacteristic phenotype of Mucopolysaccharidosis Type IVa (MPS IVA) orMorquio Syndrome. Keratan sulfate is primarily found in cartilage(joints bone growth plates, the heart valve, larynx and nasal septum)and cornea, and it is these tissues that exhibit keratan sulfateaccumulation in MPS IVA patients. Thus, forN-acetylgalactosamine-6-sulfatase (GALNS), which is deficient in MPS WAor Morquio Syndrome, it is important to show delivery of the GALNSenzyme to the growth plate of long bones, the heart valve, cornea,larynx and nose. To look at these specific tissues, which are poorlyvascularized targets, delivery of a fluorescent GALNS was investigatedin mice.

Two immunohistochemical staining methods were tested in mice: (1) humanGALNS conjugated with Alexa 488 and (2) unconjugated human GALNS. Theconjugation of human GALNS to Alexa 488 was performed using MolecularProbes Alexa Fluor 488 C₅ maleimide labeling kit (A-10254). Themaleimide conjugation chemistry resulted in a 1:1 labeling to proteinratio.

To confirm that the fluorescent tag did not interfere with uptake ofGALNS, an immunocytochemistry experiment was done using culturedsynoviocytes (ATCC #CRL-1832). A standard uptake assay was used tocompare the unconjugated GALNS with conjugated GALNS (GALNS-A488 orGALNS-A555). Cells were incubated with GALNS enzyme for 4 hours with asubsequent chase with α-L-iduronidase (IDU) for 2 hours. The resultsshowed that the Alexa 488 conjugation did not interfere with cellularuptake. FIG. 13 shows the estimated Kd for GALNS, GALNS-A488, andGALNS-A555. The uptake was measured by antigen ELISA of the cell lysaterather than enzyme activity because the labeling inactivated the enzyme.The Kd of the unconjugated and conjugated GALNS enzymes were determinedto be about equal.

To determine the stability of the fluorescent tag once the GALNS enzymewas incorporated into the cell, immunostaining on unconjugated andconjugated GALNS was performed. The primary antibody used for thestaining was a protein G-purified anti-GALNS rabbit antibody at aconcentration of 1 μg/mL. All images were taken on a Leica IRE2widefield epi-fluorescent microscope using MetaMorph software.Deconvolution of the image stacks was required to measureco-localization in these images due to the presence of out-of-planelight. The deconvolution was done using AutoQuant/AutoDeblurvisualization software using a theoretical point spread function (blindalgorithm).

The immunostaining showed fairly good overlap with signal that wasamplified over the GALNS-A488 material. The observed increase insensitivity was due to the primary and the secondary antibody both beingpolyclonal.

To determine if the GALNS enzyme was targeted to the lysosome,immunostaining of the cultured synoviocytes with Molecular ProbesLysotracker or another enzyme that localizes in the lysosome wasperformed. Lysotracker appeared to show some overlap with the GALNS-488enzyme; however, the staining wasn't uniform. A 2 hr chase withrecombinant human N-acetylgalactose amine-4-sulfatase (rhASB), alysosomal enzyme, did show some co-localization with GALNS.

The above experiments showed that GALNS-A488 enzyme is taken up by cellsand localizes to the lysosome, and can be used to determinebiodistribution in vivo.

Two in vivo studies were conducted. A first pilot study was a singledose (10 mg/kg) bolus injection in the tail vein of normal Balb/c mice,followed by a second study with multiple (5) injections every other dayof 10 mg/kg in the tail vein of normal Balb/c mice. Table 13 and Table142 describe the experimental plans for the first and second studies,respectively.

TABLE 13 Experimental Design of First Pilot Study 2 hr Time Group TotalPoint 24 hr Time Point PBS Control 4 2 2 GALNS-A488 4 2 2 UnlabeledGALNS 4 2 2 Unlabeled ASB 1 1 0

TABLE 14 Experimental Design of Second Study 2 hr 8 hr Group Total TimePoint 4 hr Time Point Time Point PBS Control 2 1 0 1 PBS/Cys Control 4 20 2 GALNS-A488 9 3 3 3 Unlabeled GALNS 6 3 0 3 Unlabeled ASB 3 2 0 1

In the first pilot study, the heart, the liver and the tibia/femur jointwere harvested at 2 hour and 24 hour time points. In the second study,the heart, the kidney, the liver, and the bone with quadricep and soleuswere harvested at 2 hour, 4 hour and 8 hour time points. For bothstudies, the heart, kidney, and liver were immersion fixed in 4%paraformaldehyde (PFA) for 4 days, paraffin embedded, then sectioned to7 μm thickness. The bone, including the muscle in the second study, wasimmersion fixed in 4% PFA for 8 days, decalcified, paraffin embedded,and sectioned to 7 μm thickness.

Images of the GALNS-A488 injected mice were acquired on a Zeiss laserscanning confocal microscope. For the analysis in the first pilot study,one confocal stack per sample was acquired for the heart valve and liverand used for volumetric analysis. Two confocal stacks/sample wereacquired for the growth plate and used for volumetric analysis. In thesecond study, one confocal stack/sample for heart valve, kidney andliver was acquired and used for volumetric analysis; two confocalstacks/sample for growth plate and zone of rest cartilage (zrc) wereacquired and used for volumetric analysis.

The conclusions from the confocal microscopy imaging studies were: (1)it was possible to detect fluorescent GALNS in vivo; (2) the signal wasspecific (absence of background) and the localization was lysosomal; (3)the presence of GALNS was demonstrated in the sinusoidal cell in theliver; (5) in the heart, the GALNS enzyme was present in the septum andthe atrium, but more importantly it was clearly visible at the level ofthe heart valve, where it was more deeply distributed after multipleinjections; (6) at the femur/tibia junction, the GALNS enzyme waspresent in the mineralized part of the bone (epiphysis), as well as themarrow. GALNS was present in the growth plate. More particularly, GALNSwas abundant in the chondrocytes of the resting zone (or zone of reservecartilage), present at the beginning of the proliferative zone, andreappeared abundantly in the ossification zone at the end of the growthplate. Although difficult to quantify the cumulative effect of multipleinjections, the second study seemed to display a broader distribution.Table 15 shows a summary of the confocal microscopy imaging studies.

TABLE 15 Biodistribution of GALNS in Mice Tissue Localization Bone(Femur) Mineralized region Yes Bone Marrow Yes Growth Plate Yes HeartHeart valve Yes Atrium Yes Septum Yes Liver Hepatocyte No SinusoidalCell Yes

For secondary staining, the initial step was optimization of the GALNSprimary antibody. Various tissues were stained with dilutions of 1:100to 1:400 with the protein G-purified anti-GALNS rabbit antibody. Resultsin the first pilot study indicated that a dilution of 1:100 was optimalfor a high signal to noise ratio. This result was confirmed in thesecond study. The remaining slides were processed at a primary antibodydilution of 1:100 and a secondary antibody dilution of 1:1000.

Signal for Balb/c mice dosed with GALNS had a signal above control(i.e., PBS-Cys dosed mice) when stained with the protein G-purifiedanti-GALNS antibody. To confirm that the GALNS enzyme was localized inthe lysosome, the sections were stained with an anti-LAMP1 antibody.LAMP1 is a marker for lysosomes. The images showed overlap between theanti-LAMP1 and anti-GALNS antibodies, indicating that the GALNS enzymewas localized in the lysosome.

Overall, the two in vivo studies indicate that GALNS biodistribution islinked to vascularization, i.e., the more vascularized tissues containmore fluorescent signal. More importantly, the studies demonstrate thepresence of GALNS at the sites of keratan sulfate accumulation inMorquio Syndrome, even if these sites are poorly vascularized.

Example X Effects of Recombinant Human N-Acetylgalactosamine-6-Sulfatase(GALNS) and Other Lysosomal Sulfatase Enzymes in Mice Deficient inLysosomal Sulfatase Enzyme Activity

The effects of the active highly phosphorylated human lysosomalsulfatase enzymes of the invention, e.g., recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS), in mice deficient inlysosomal sulfatase enzyme activity are evaluated.

The recombinant human GALNS protein is expressed in G71S cells andpurified. Other recombinant human lysosomal sulfatase enzymes can beexpressed and purified basically according to methods described hereinor by procedures known in the art.

Several mouse models of human lysosomal sulfatase enzyme deficiency havebeen described, including: Metachromatic Leukodystrophy (MLD)(arylsulfatase A deficiency), (Hess et al., Proc. Natl. Acad. Sci. USA93:14821-14826, 1996), Mucopolysaccharidosis type VI (MPS VI) orMaroteaux-Lamy syndrome (arylsulfatase B deficiency) (Evers et al.,Proc. Natl. Acad. Sci. USA 93:8214-8219, 1996), Mucopolysaccharidosistype II (MPS II) or Hunter syndrome (iduronate-2-sulfatase deficiency)(Muenzer et al., Acta Paediatr. Suppl. 91(439):98-99, 2002; Cardone etal., Hum. Mol. Genet. 15:1225-1236, 2006), Mucopolysaccharidosis typeIIIa (MPS IIIa) or Sanfilippo A syndrome(sulfamidase/heparan-N-sulfatase deficiency) (Bhaumik et al.,Glycobiology 9(12):1389-1396, 1999), Mucopolysaccharidosis type IVa (MPSIVa) or Morquio A syndrome (N-acetylgalactosamine-6-sulfatasedeficiency) (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003), andMultiple Sulfatase Deficiency (MSD) (sulfatase modifying factor 1deficiency) (Settembre et al., Proc. Natl. Acad. Sci. USA 104:4506-4511,2007). A mouse model of Mucopolysaccharidosis type IIId (MPS IIId) orSanfilippo D syndrome (N-acetylglucosamine-6-sulfatase deficiency) hasyet to be described.

Mouse models of human lysosomal sulfatase enzyme deficiency can be usedto assess the feasibility of enzyme replacement therapy (ERT) as a meansfor treating lysosomal storage disorders. For example, MPS IVa knock-outmice (GALNS^(−/−) mice; Tomatsu et al., Hum. Mol. Genet. 12:3349-3358,2003) have no detectable GALNS enzyme activity and display increasedurinary glycosaminoglycans (GAGs), i.e., keratin sulfate andchondroitin-6-sulfate, and accumulation of GAGs in multiple tissues andorgans, e.g., liver, kidney, spleen, heart, brain, bone marrow andcartilage. The GALNS^(−/−) mice do not, however, display skeletalabnormalities associated with the human disease. Another MPS IVa mousemodel was developed that expresses an inactive human GALNS and amutated, inactive endogenous mouse GALNS (GALNS^(tm(hC79S.mC76S)slu)mice; Tomatsu et al., Hum. Mol. Genet. 14:3321-3335, 2005). InGALNS^(tm(hC79S.mC76S)slu) mice, which have no detectable GALNS enzymeactivity, urinary GAG excretion is increased, GAGs accumulate inmultiple tissues, including visceral organs, brain, cornea, bone,ligament and bone marrow, lysosomal storage is marked in multipletissues, and bone storage is evident. The pathological alterations inGALNS^(tm(hC79S.mC76S)slu) mice are different from those observed inGALNS^(−/−) mice. However, like the GALNS^(−/−) mice,GALNS^(tm(hC79S.mC76S)slu) mice do not display skeletal abnormalitiesassociated with the human disease. Thus, GALNS^(−/−) orGALNS^(tm(hC79S.mC76S)slu) mice can be used to investigate the effect ofadministration of recombinant human GALNS on increased urinary GAGs andaccumulation of GAGs in the tissues.

Four week old GALNS^(−/−), GALNS^(tm(hC79S.mC76S)slu) or wild-type miceare given weekly intravenous injections (n=at least 6 or 8 per group) ofvarious doses of recombinant human GALNS (e.g., 0.1, 0.3, 1, 3, 10mg/kg) or a vehicle control through 16-20 weeks of age, and thensacrificed for histological examination. Urine is collected from miceand urinary GAG excretion is determined as described (Tomatsu et al.,Hum. Mol. Genet. 12:3349-3358, 2003). Pathological examination ofvarious tissues is performed as described (Tomatsu et al., Hum. Mol.Genet. 12:3349-3358, 2003).

Using the GALNS^(−/−) or GALNS^(tm(hC79S.mC76S)slu) mice, therecombinant human GALNS of the invention is expected to demonstrate theability to reduce: (1) urinary GAG excretion; (2) accumulation of GAGsin multiple tissues, e.g., visceral organs, brain, cornea, bone,ligament and bone marrow; (3) lysosomal storage in multiple tissues; and(4) bone storage.

The effect of recombinant human GALNS is investigated in a mouse modelof Multiple Sulfatase Deficiency (MSD) (SUMF1^(−/−) mice; Settembre etal., Proc. Natl. Acad. Sci. USA 104:4506-4511, 2007). BecauseSUMF1^(−/−) mice display frequent mortality early in life, injections ofthese mice with recombinant human GALNS is initiated earlier than thatdescribed above for GALNS^(−/−) mice.

Following procedures known in the art, the effects of other recombinanthuman lysosomal sulfatase enzymes, i.e., arylsulfatase A, arylsulfataseB, iduronate-2-sulfatase, sulfamidase/heparan-N-sulfatase, andN-acetylglucosamine-6-sulfatase, are investigated in mouse models of MLD(ASA^(−/−) mice; Hess et al., Proc. Natl. Acad. Sci. USA 93:14821-14826,1996), MPS VI (Asl-s^(−/−) mice; Evers et al., Proc. Natl. Acad. Sci.USA 93:8214-8219, 1996), MPS II (ids^(y/−) mice; Cardone et al., Hum.Mol. Genet. 15:1225-1236, 2006), MPS IIIa (Bhaumik et al., Glycobiology9(12):1389-1396, 1999) and MSD (SUMF1^(−/−) mice; Settembre et al.,Proc. Natl. Acad. Sci. USA 104:4506-4511, 2007).

Example XI Treatment of Human Patients with Mucopolysaccharidis Type IVA(or Morquio Syndrome) or Other Lysosomal Sulfatase Enzyme Deficiencieswith Recombinant Human N-Acetylgalactosamine-6-Sulfatase (GALNS) andOther Lysosomal Sulfatase Enzymes

Human patients manifesting a clinical phenotype of lysosomal sulfataseenzyme deficiency, such as in patients diagnosed withMucopolysaccharidosis Type IVA (MPS IVa or Morquio Syndrome), arecontemplated for enzyme replacement therapy with the recombinant enzyme,i.e., human N-acetylgalactosamine-6-sulfatase (GALNS). All patientssuffering from a lysosomal sulfatase enzyme deficiency manifest someclinical evidence of excessive or harmful visceral and soft tissueaccumulation of storage material in their lysosomes as manifested byvarying degrees of functional impairment or worsening health statusassociated with a particular lysosomal storage disease. All the MPS IVapatients manifest some clinical evidence of bone deformity, shortstature and abnormal gait, and/or accumulation of glycosaminoglycan(GAG) in the blood or urine, with varying degrees of functionalimpairment.

Preferably, enzyme levels are monitored in a patient suffering from alysosomal sulfatase enzyme deficiency to confirm the absence or reducedactivity of the lysosomal sulfatase enzyme in their tissues. Patientswith less than 10%, preferably less than 5%, more preferably less than2% and even more preferably less than 1% of the lysosomal enzymeactivity in an otherwise normal subject are suitable candidates fortreatment with the appropriate lysosomal sulfatase enzyme. Data may becollected to determine the patient's lysosomal sulfatase enzyme activitybefore, during and after therapy.

Efficacy is determined by measuring the percentage reduction in urinaryexcretion of the substrate, i.e., glycosaminoglycan (GAG) of thelysosomal sulfatase enzyme over time. The urinary GAG levels in patientssuffering from a lysosomal sulfatase enzyme deficiency are compared tonormal excretion levels and/or levels in untreated patients sufferingfrom the same lysosomal sulfatase enzyme deficiency and/or levels in thesame patient before therapy with the lysosomal sulfatase enzyme. Agreater than 25% reduction, preferably greater than 50% reduction, inexcretion of undegraded GAGs following therapy with the lysosomalsulfatase enzyme is a valid means to measure an individual's response totherapy.

Efficacy can also be determined according to the reduced signs andsymptoms of pathology associated with the lysosomal storage disease.Efficacy can be determined by tissue biopsy and examination of cellsand/or lysosomes to determine the extent by which GAGs have been reducedin the lysosomes, cells or tissues. Efficacy can be determined byfunctional assessments, which may be objective or subjective (e.g.,reduced pain or difficulty in function, increased muscle strength orstamina, increased cardiac output, exercise endurance, changes in bodymass, height or appearance, and the like).

A pharmaceutical composition comprising recombinant human GALNS,expressed in G71S cells and purified, and formulated according toprocedures known in the art. It is preferred to administer thepharmaceutical compositions of the invention intravenously.

The basic design of an initial clinical study to investigate the effectof administration of recombinant human GALNS to MPS IVa patientsinvolves an open label, dose escalation safety/efficacy study in whichvarious doses of enzyme are administered intravenously to the patientsat a fixed interval, for example and not for limitation, weekly enzymeinjections.

For MPS IVa patients, efficacy is determined by measuring, for example,decreased blood or urinary GAG, which is likely to be observed withinweeks of ERT, increased endurance in tests of cardiac, pulmonary and/ormotor function, which is likely to be observed within months of ERT,and/or skeletal changes and/or body growth, which is likely to beobserved within years of ERT.

Urinary GAG measurements are useful for establishing an appropriate doseregimen, as well as for determining efficacy, by measuring thepercentage reduction in urinary GAG excretion over time.

A variety of endurance tests may be employed, including for example andnot for limitation, walk tests (distance walked in 6 or 12 minutes),stair climb (stairs per minute), and pulmonary/respiratory function,including cardiac function (ECG, echocardiogram), pulmonary function(FVC, FEV₁, peak flow).

For younger patients undergoing treatment for extended periods of time,growth (height) may be measured.

The lysosomal storage diseases associated with deficiency in lysosomalsulfatase enzyme activity that can be treated or prevented using themethods of the present invention are: Metachromatic Leukodystrophy(MLD), Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamysyndrome, Mucopolysaccharidosis type II (MPS II) or Hunter syndrome,Mucopolysaccharidosis type IIIa (MPS IIIa) or Sanfilippo A syndrome,Mucopolysaccharidosis type IIId (MPS IIId) or Sanfilippo D syndrome,Mucopolysaccharidosis type IVa (MPS IVa) or Morquio A syndrome, orMultiple Sulfatase Deficiency (MSD). For each lysosomal storage disease,the recombinant lysosomal sulfatase enzyme would comprise a specificlysosomal sulfatase enzyme.

For methods involving MLD, the preferred lysosomal sulfatase enzyme isarylsulfatase A. For methods involving MPS VI, the preferred lysosomalsulfatase enzyme is arylsulfatase B. For methods involving MPS II, thepreferred lysosomal sulfatase enzyme is iduronate-2-sulfatase. Formethods involving MPS IIIA, the preferred lysosomal sulfatase enzyme issulfamidase/heparan-N-sulfatase. For methods involving MPS IIID, thepreferred lysosomal sulfatase enzyme is N-acetylglucosamine-6-sulfatase.For methods involving MPS IVA, the preferred lysosomal sulfatase enzymeis N-acetylgalactosamine-6-sulfatase. For methods involving MSD, thepreferred lysosomal sulfatase enzyme isN-acetylgalactosamine-6-sulfatase.

Numerous modifications and variations of the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

1. A purified preparation of recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) enzyme, said GALNS enzymecomprising an amino acid sequence at least 95% identical to amino acids27 to 522 of SEQ ID NO: 4, and (a) having a purity of at least about 95%as determined by Coomassie Blue staining when subjected to SDS-PAGEunder non-reducing conditions, (b) having at least about 90% conversionof the cysteine residue at position 53 to C_(α)-formylglycine (FGly),and (c) having between 0.5 to 0.8 bis-phosphorylated oligomannose chainsper monomeric protein chain, wherein at least 70% of said GALNS enzymeis in a precursor form as determined by Coomassie Blue staining whensubjected to SDS-PAGE under reducing conditions.
 2. A sterilecomposition comprising (a) a recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) enzyme that comprises an aminoacid sequence at least 95% identical to amino acids 27 to 522 of SEQ IDNO: 4, said GALNS enzyme (i) having a purity of at least about 95% asdetermined by Coomassie Blue staining when subjected to SDS-PAGE undernon-reducing conditions, (ii) having at least about 90% conversion ofthe cysteine residue at position 53 to C_(α)-formylglycine (FGly), and(iii) having between 0.5 and 0.8 bis-phosphorylated oligomannose chainsper monomeric protein chain, wherein at least 70% of said GALNS enzymeis in a precursor form as determined by Coomassie Blue staining whensubjected to SDS-PAGE under reducing conditions; and (b) a sterilepharmaceutically acceptable diluent.
 3. The purified preparation ofclaim 1, wherein the GALNS enzyme is at least 95% pure as determined byRP-HPLC.
 4. The purified preparation of claim 1, wherein at least 80% ofthe GALNS enzyme is in the precursor form as determined by CoomassieBlue staining when subjected to SDS-PAGE under reducing conditions. 5.The purified preparation of claim 1, wherein at least 80% of the GALNSenzyme is in the precursor form as determined by SDS-capillary gelelectrophoresis.
 6. The purified preparation of claim 1, wherein between50% to 80% of the GALNS enzyme binds to a mannose-6-phosphate receptorcolumn.
 7. The purified preparation of claim 1, wherein the GALNS enzymeexhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to5 nM.
 8. The purified preparation of claim 1, wherein the GALNS enzymeexhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to3.5 nM.
 9. The sterile composition of claim 2, wherein the GALNS enzymeis at least 95% pure as determined by RP-HPLC.
 10. The sterilecomposition of claim 2, wherein at least 80% of the GALNS enzyme is inthe precursor form as determined by SDS-capillary gel electrophoresis.11. The sterile composition of claim 2, wherein at least 90% of theGALNS enzyme is in the precursor form as determined by SDS-capillary gelelectrophoresis.
 12. The sterile composition of claim 2, wherein between50% to 80% of the GALNS enzyme binds to a mannose-6-phosphate receptorcolumn.
 13. The sterile composition of claim 2, wherein the GALNS enzymeexhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to5 nM.
 14. The sterile composition of claim 2, wherein the GALNS enzymeexhibits a specific uptake (Kuptake) into fibroblasts that is about 1 to3.5 nM.
 15. A purified preparation of recombinant humanN-acetylgalactosamine-6-sulfatase (GALNS) enzyme, said GALNS enzymecomprising an amino acid sequence at least 95% identical to amino acids27 to 522 of SEQ ID NO: 4, and (a) having a purity of at least about 95%as determined by Coomassie Blue staining when subjected to SDS-PAGEunder non-reducing conditions, (b) having at least about 90% conversionof the cysteine residue at position 53 to C_(α)-formylglycine (FGly),and (c) having between 0.5 to 0.8 bis-phosphorylated oligomannose chainsper monomeric protein chain, wherein at least 70% of said GALNS enzymeis in a precursor form as determined by SDS/PAGE Coomassie blue underreducing conditions, and wherein said GALNS enzyme exhibits a specificuptake (Kuptake) into fibroblasts that is about 1 to 5 nM.
 16. Thepurified preparation of claim 15, wherein the GALNS enzyme is at least95% pure as determined by RP-HPLC.
 17. The purified preparation of claim15, wherein at least 80% of the GALNS enzyme is in the precursor form asdetermined by Coomassie Blue staining when subjected to SDS-PAGE underreducing conditions.
 18. The purified preparation of claim 15, whereinat least 80% of the GALNS enzyme is in the precursor form as determinedby SDS-capillary gel electrophoresis.
 19. The purified preparation ofclaim 15, wherein at least 90% of the GALNS enzyme is in the precursorform as determined by SDS-capillary gel electrophoresis.
 20. Thepurified preparation of claim 15, wherein the GALNS enzyme exhibits aspecific uptake (Kuptake) into fibroblasts that is about 1 to 3.5 nM.